UNIVERSITÀ DEGLI STUDI DI MILANO
FACOLTÀ DI AGRARIA
Graduate School in Molecular Sciences and Plant, Food and
Environmental Biotechnology
PhD programme in Food Science, Technology and
Biotechnology
XXVII cycle
Bacterial enzymatic activities as potential
markers for assessing the
technological properties of
(un)processed milk
Scientific field AGR/15
MARILU` DECIMO
Tutor: Prof. Ivano De Noni
Co-tutor: Dr. Milena Brasca
PhD Coordinator: Prof. Maria Grazia Fortina
2013/2014
To my parents and
my grandmother Pina
Index
INDEX
0 PREFACE
0.1 Milk and sources of microbial contamination
0.2 Psychrotrophic bacteria
0.3 Enzymes in milk
0.3.1 Indigenous milk enzymes
0.3.2 Bacterial extracellular enzymes
0.3.2.1 Proteases
0.3.2.2 Lipases and Phospholipases
0.4 Control of psychrotrophs and related enzymes
0.5 References
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15
16
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1 STATE OF THE ART
1.1 References
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24
2 AIMS OF THE STUDY
25
3 RESULTS AND DISCUSSION
3.1 Characterization of Gram-negative psychrotrophic bacteria
3.2 Material and methods
3.2.1 Psychrotrophic bacterial counts and isolation of gram-negative
psychrotrophic (GNP) strains
3.2.2 Phenotypic characterization and biochemical tests
3.2.3 DNA extraction
3.2.4 Identification of psychrotrophic isolates by 16S rRNA and rpoB
genes sequencing
3.2.5 Randomly amplified polymorphic DNA (RAPD) analysis
3.2.6 Detection of the aprX gene in psychrotrophic strains (aprX –
PCR)
3.3 Results and discussion
3.3.1 Psychrotrophic bacterial counts and isolation of gram-negative
psychrotrophic (GNP) strains
3.3.2 Phenotypic properties
3.3.3 Growth and enzymatic activity at different temperatures
3.3.4 Assessment of aprX gene in psychrotrophic strains
3.4 Conclusions
3.5 References
26
26
27
3.6 Characterization of volatile compounds in milk contaminated with
psychrotrophic bacteria by SPME gas chromatography-mass
spectrometry
3.7 Material and methods
3.7.1 Bacterial strains and milk contamination
3.7.2 VOC determination by SPME/GC-MS
3.7.3 Sensory evaluation
3.8 Results and discussion
3.8.1 VOC determination of spoiled UHT milk samples during storage
3.8.2 Sensory analysis of microbially spoiled milk samples
3.9 Conclusions
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48
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59
Index
3.10 References
59
3.11 Fatty acids released from cream by psychrotrophs
3.12 Material and methods
3.12.1 Microorganisms
3.12.2 Physico-chemical analysis of cream
3.12.3 Preparation of partially purified extracellular lipase
3.12.4 Changes in the substrate specificity of crude enzyme
3.12.5 Preparation of spoiled cream samples
3.12.6 Free fatty acid analysis
3.13 Results and discussion
3.13.1 Physico-chemical characteristics of cream
3.13.2 Lipolytic activity of partially purified lipases
3.13.3 Release of FFAs by psychrotrophs
3.14 Conclusions
3.15 References
64
65
65
65
65
65
66
66
67
67
67
71
73
74
3.16 Proteomic characterization of extracellular proteases of P.
fluorescens strains
3.17 Material and methods
3.17.1 Bacterial strains and culture conditions
3.17.2 Preparation of crude enzyme extract
3.17.3 Casein zymography and detection of extracellular caseinolytic
activity
3.17.4 Proteomic analysis of crude enzyme extract from PS19 P.
fluorescens strain
3.17.5 In gel digestion
3.17.6 In solution digestion
3.17.7 NanoLC–ESI-MS/MS peptide analysis
3.17.8 Mass spectral data elaboration and database searching
3.18 Results and discussion
3.18.1 Extracellular caseinolytic potential of P. fluorescens PS19,
PS60 and PS24
3.18.2 NanoLC–ESI-MS/MS and identification of P. fluorescens PS19
extracellular proteases
3.19 Conclusions
3.20 References
3.21 Identification of peptides from hydrolysis of casein fractions by P.
fluorescens PS19 enzyme extract
3.22 Material and methods
3.22.1 Hydrolysis of casein fractions by enzyme extract from P.
fluorescens PS19
3.22.2 Reversed-phase – High performance liquid chromatography
(RP-HPLC)
3.23 Results and discussion
3.24 References
Appendix 1. COPIES OF ABSTRACTS OF PAPERS, ORAL
COMMUNICATIONS AND POSTERS
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Index
Appendix 2. INDEX OF TABLES
Appendix 3. INDEX OF FIGURES
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100
ABSTRACT
Psychrotrophic bacteria are responsible for the highest spoilage of unprocessed or heated milk
during storage because of their capacity to synthesize thermostable extracellular proteases and
lipases. The activities of these enzymes lead to formation of off-odours/flavours, gelation of
milk, lowering of milk foaming properties, loss of sensory quality and shortening of the shelf
life. To date, still little is known about the specific proteolytic and lipolytic pathways of these
thermostable enzymes.
Initially we evaluated the enzymatic traits of 80 raw milk-associated psychrotrophic strains.
Among psychrotrophic isolates, Pseudomonas were the most commonly occurring
contaminants (78.75%) being Pseudomonas fluorescens the predominant isolated species (30.16
%), along with Enterobacteriaceae (21.25%), primarily Serratia marcescens (52.94 %). Fortyone of the psychrotrophic strains were positive for all the enzymatic activities. The highest
number of positive strains for all incubation temperatures was found for the lipolytic activity
(59), followed by proteolytic (31) and lecithinase (28) activities. The enzymatic traits varied
among the Pseudomonas and Enterobacteriaceae strains and were markedly influenced by
incubation temperature being 30 °C the optimal one. The aprX gene was detected in 19 out of
80 psychrotrohic strains and it resulted widespread among P. fluorescens strains (15 out of 18).
The second part of the research was focused on the evaluation of spoilage potential of
psychrotrophic strains by analyzing the production of volatile organic compounds (VOCs) and
the release of free fatty acids (FFAs). From results of SPME-GC/MS analysis, different species
of the genus Pseudomonas and Serratia marcescens produced a complex and strain-dependent
VOCs profiles in UHT milk samples at different storage and time conditions. Fifty-six VOCs
belonging to 7 chemical groups (aldehydes, ketones, fatty acids, esters, alcohols, sulphur
compounds and hydrocarbons) were identified. Generally, the VOCs went to increase during
the storage time both in the control and contaminated milk samples, some compounds being
detected only in the latter samples. Compounds such as 3-methylbutan-2-ol, 3-methylhexan-2ol, pentan-1-ol and 3,3-dimethylhexane were detectable only for P. fragi. P. rhodesiae was the
only species producing pentane-2,3-dione, heptane and 3-methylhexane while hexane was
released only by P. fluorescens. P. mosselii and P. fragi produced the highest number of
sulphur compounds and alcohols, respectively. The highest number of FFAs and ketons was
detected in the headspace of milk samples contaminated by P. rhodesiae and S. marcescens. P.
fluorescens provided the lowest development of VOCs. 3-methylbutan-1-ol, 2 methylpropan-1ol, 3-hydroxybutan-2-one, butane-2,3-dione and butanoic and hexanoic acids could be regarded
as potential markers of psycrotrophic contamination useful for the early detection of milk
bacterial spoilage. Regarding the release of FFAs, different quantities of these compounds have
been released from milk fat by tested bacteria, between and within species, in relation to diverse
capacity for production of lipolytic enzymes. Palmitic (16:0), oleic (18:1) and linoleic (18:2)
acids levels were found to be the highest among the SFAs, MUFAs and PUFAs, respectively. P.
fluorescens PS73 and P. fluorescens PS81 were the major FFAs producers, at 24 h and 4 days
of incubation, respectively while H. alvei PS57 and P. fragi PS55 were the less active in lipid
breakdown at both the incubation conditions.
Lipases from psychrotrophic strains showed a variable range of specificity toward fatty acid
esters with different fatty acid chain lengths, being P. fragi PS55, P. putida PS17, P.
fluorescens PS14 and P. fulva PS10 the more active to hydrolyse triglycerides. Lipase from P.
rhodesiae PS62 showed the highest hydrolytic resistance toward all tested fatty acid
triglycerides.
8
Finally, proteomic characterization of extracellular proteases of P. fluorescens strains has been
performed. One thermostable protease of approximately 45 kDa was detected in each of the
cell-free supernatant of the selected strains on a casein zymogram gel. After concentration by
ultrafiltration (10 kDa), the protease extract of P. fluorescens PS19 showed a high proteolytic
activity and two additional proteolytic bands with molecular masses of approximately 15 and 25
kDa on casein zymography. This extract was subjected to proteomic characterization by
nLC/MS/MS analysis of both in gel and in solution digestion. Results showed the protease of 45
kDa to correspond to P. fluorescens AprX metalloprotease (acc. no. C9WKP6, UniProt). In
addition, the same results leaded to recognize the 15 kDa protease as a fragment of this AprX
metalloprotease. On the contrary, the 25 kDa protease showed no homology to any known
protein of Pseudomonas spp.
The characterization by LC/MS of the peptides profile generated by the action of thermostable
proteases of the same strain on milk caseins is still under investigation.
Overall, this study provides a better understanding of the enzymatic activities of psychrotrophic
bacteria in milk.
9
RIASSUNTO
I batteri psicrotrofi sono i principali responsabili del deterioramento del latte crudo o
termotrattato poiché sono in grado di sintetizzare proteasi e lipasi extracellulari termostabili,
causa di formazione di odori e sapori sgradevoli, fenomeni di gelificazione, riduzione delle
proprietà schiumogene del latte, perdita di qualità sensoriale e riduzione della shelf-life. Ad
oggi, non esiste ancora una sufficiente conoscenza degli specifici pathways proteolitici e
lipolitici di questi enzimi termostabili.
Inizialmente questo lavoro ha riguardato la caratterizzazione dell’attività enzimatica di 80 ceppi
di psicrotrofi isolati da latte crudo. I batteri appartenenti al genere Pseudomonas sono stati i più
isolati (78.75%) e Pseudomonas fluorescens è risultata la specie predominante (30.16%); tra le
Enterobacteriaceae (21.25%), Serratia marcescens è stata la specie più frequentemente isolata
(52.94%). Quarantuno ceppi di psicrotrofi mostravano tutte le attività enzimatiche. Il più alto
numero di ceppi positivi a tutte le temperature di incubazione è stato osservato per l’attività
lipolitica (59) e, a seguire, proteolitica (31) e lecitinasica; i tratti enzimatici variavano tra i ceppi
di Pseudomonas e Enterobacteriaceae ed erano marcatamente influenzati dalla temperature di
incubazione, essendo 30 °C quella ottimale. Il gene aprX è stato ritrovato in 19 ceppi di
Pseudomonas ed è risultato esser diffuso tra i ceppi di P. fluorescens (15 su18).
La seconda parte della ricerca è stata focalizzata sulla valutazione della produzione di composti
organici volatili (VOCs) e del rilascio di acidi grassi liberi (FFAs) da ceppi di batteri psicrotrofi.
Diverse specie del genere Pseudomonas e il ceppo Serratia marcescens hanno mostrato profili
di VOCs complessi e dipendenti dal ceppo batterico inoculato in campioni di latte UHT,
durante le differenti condizioni di incubazione. In particolare, sono stati identificati 56 VOCs
appartenenti a aldeidi, chetoni, acidi grassi, alcoli, composti solforati e idrocarburi.
Generalmente, il numero di VOCs tendeva ad incrementare con il progredire del tempo di
incubazione, sia nel latte controllo non inoculato sia nei campioni di latte contaminati. Tra i
VOCs rilevati, alcune molecole sono state individuate solo quando il latte era contaminato da
uno specifico ceppo microbico. Nel dettaglio, 3-metilbutan-2-olo, 3-metilesan-2-olo, pentan-1olo e 3,3-dimetilesano sono stati rilevati solo a seguito dello sviluppo di P. fragi. P. rhodesiae è
stata l’unica specie a produrre pentano-2,3-dione, eptano e 3-metilesano, mentre l’esano è stato
rilasciato solo in campioni di latte contaminati con P. fluorescens. La maggior produzione di
composti solforati e alcoli è stata individuata nello spazio di testa del latte contaminato con P.
mosselii e P. fragi, rispettivamente. Lo sviluppo e l’attività di P. rhodesiae e S. marcescens
sono risultati associati ad un più alto numero di acidi grassi e chetoni mentre P. fluorescens ha
mostrato la più scarsa produzione di composti volatili. Alcuni VOCs come 3-metilbutan-1-olo,
2-metilpropan-1-olo, 3-idrossibutan-2-one, butano-2,3-dione, acido butanoico ed acido esanoico
potrebbero perciò rappresentare potenziali marker per il riconoscimento dell’attività enzimatica
di batteri psicrotrofi e per la precoce individuazione del deterioramento del prodotto.
Per quanto riguarda il rilascio di FFAs, diverse quantità di questi composti sono state rilasciate
dai batteri psicrotrofi appartenenti a specie diverse e alla stessa specie conseguentemente alla
diversa capacità di produrre lipasi. Gli acidi palmitico (16:0), oleico (18:1) e linoleico (18:2)
sono risultati i più presenti tra gli acidi grassi saturi, monoinsaturi e polinsaturi. P. fluorescens
PS73 e P. fluorescens PS81 sono state le specie che hanno prodotto la maggior quantità di
FFAs, a 24 h e 4 giorni di incubazione, rispettivamente. Al contrario, H. alvei PS57 e P. fragi
PS55 hanno rilasciato la minor quantità di FFAs ad entrambi i tempi di incubazione.
Le lipasi dei ceppi di psicrotrofi hanno mostrato una specificità variabile nei riguardi degli
esteri degli acidi grassi con diversa lunghezza della catena carboniosa. P. fragi PS55, P. putida
PS17, P. fluorescens PS14 and P. fulva PS10 sono risultate le specie più attive nell’idrolisi dei
10
trigliceridi. La lipasi del ceppo di P. rhodesiae PS62 ha mostrato la più scarsa capacità idrolitica
verso tutti i trigliceridi testati.
Infine, è stata effettuata la caratterizzazione proteomica di proteasi extracellulari di ceppi di P.
fluorescens. Una proteasi termostabile di circa 45 kDa è stata individuata su casein zymogram
gel in ciascun surnatante dei ceppi batterici selezionati. L’estratto enzimatico del ceppo P.
fluorescens PS19, concentrato per ultrafiltrazione (10 kDa), ha mostrato un’elevata attività
proteolitica e due ulteriori bande proteolitiche di circa 15 e 25 kDa. I risultati delle analisi
nLC/MS/MS dopo digestione sia in gel che in solution hanno evidenziato che la proteasi di 45
kDa corrisponde ad una AprX metalloproteasi di P. fluorescens (acc. no. C9WKP6, UniProt).
La proteasi di 15 kDa è stata riconosciuta come un frammento della stessa AprX
metalloproteasi, mentre la proteasi di 25 kDa non ha mostrato nessuna omologia con alcuna
delle proteine note di Pseudomonas.
La caratterizzazione tramite LC/MS del profilo peptidico generato dall’azione delle proteasi
termostabili dello stesso ceppo sulle frazioni caseiniche del latte è in corso di studio.
In generale, questo studio fornisce ulteriori conoscenze per la lo studio delle attività
enzimatiche di batteri psicrotrofi nel latte.
11
Preface
0. PREFACE
0.1 Milk and sources of microbial contamination
Raw milk, due to its high nutritional value, water content and near neutral pH, is an
environment that contains a diverse and complex microbial population (Quigley et al., 2011;
Vacheyrou et al., 2011). The microbial composition of raw milk is variable according to a
variety of sources, including the teat apex, milking equipment, air, water, feed, grass, soil, and
other environments (Lejeune & Rajala-Schultz 2009; Vacheyrou et al., 2011).
The mammary glands of very young cows yield no bacteria in aseptically collected milk
samples, but as numbers of lactations increase, so do the chances of isolating bacteria in milk
drawn aseptically from the teats. The stresses placed on the cow’s teats and mammary glands by
the very large amounts of milk produced and the actions of the milking machine cause teat
canals to become more open and teat ends to become misshapen as time passes. These stresses
may open the teat canal for the entry of bacteria capable of infecting the glands and
consequently to enter in milk. (Ledenbach & Marshall, 2009). Microbial populations
contaminating the raw milk can include bacteria of technological relevance such as lactic acid
bacteria that are fundamental if the milk is employed in cheese-making (Coppola et al., 2008);
however, spoilage bacteria including both Gram-positive and Gram-negative microorganisms
occur sometimes with considerable effects on the quality of milk and derived products (Cousin,
1982).
Contamination of milk by spoilage microorganisms also occurs due to inadequately sanitized
surfaces of milking and milk storage equipment. The organisms grow in milk residues present
in crevices, joints, rubber gaskets, and dead ends of badly cleaned milking plants. Although
many different bacterial types can be introduced into milk from milk mineral deposits present in
milking equipment, the most important of these are the Gram-negative psychrotrophs, which
predominate among the microflora that adhere to stainless-steel milk transfer pipelines.
Ancillary equipment such as agitators, dipsticks, outlet plugs, and cocks can be difficult to clean
and these may be a possible source of contamination (Mac Phee & Griffiths, 2002).
The combination of longer storage times and lower temperatures of raw milk throughout the
dairy chain prior to heat treatment creates selective conditions for the growth of psychrotrophic
bacteria, particularly Pseudomonas spp., altering the structural and sensory properties of the
finished dairy product (Chen et al., 2003; Lafarge et al., 2004). Milk may be collected from
farms on alternate days, or even longer in some instances. Thus, at collection, part of the milk in
the bulk tank may be 48 h old or more. Although alternate-day collection may have little effect
on the bacteriological quality of milk rapidly cooled to 4 °C or below before addition to the
tank, the growth potential of the raw milk microflora is significantly affected. Thus, milk
collected on alternate days will contain a greater number of bacteria that are entering the
exponential phase of growth when the milk arrives at the processing site, and the amount of
time that this milk can be subsequently stored will be reduced. For example, it has been shown
that Pseudomonas spp. isolated from milk that had been stored at 7 °C for 3 days grew 10 times
faster at this temperature, had 1000-fold more proteolytic activity, and were 280-fold more
lipolytic than pseudomonads isolated from freshly drawn milk. Moreover, changes in dairy
industry practices, such as the introduction of a 5-day working week and milk shortages at
certain times of the year due to the adoption of quota systems, have led to milk being stored for
longer times before processing. Thus, the temperature at which milk is stored becomes critical.
It has been recommended that milk is cooled to, and maintained at, 3 °C on receipt at the
processing plant before storage. When the milks were stored for a further 48 h at 6 °C, the
psychrotroph count increased by 2 log cycles to 1.3×107 cfu ml-1. The majority of bacteria
12
Preface
present were pseudomonads (70.2%) but Enterobacteriaceae (7.7%), Gram-positive bacteria
(6.9%), and other Gram-negative, rod-shaped organisms were also isolated.
Milk is usually transported in insulated tanks or in refrigerated tankers, and may be transferred
to larger vehicles for longer journeys. During transportation, the main cause of increased
bacterial count is inadequately cleaned vehicles and growth of bacteria already present in the
milk. The latter is dependent on the milk temperature and journey time. A twofold increase in
count is common during transportation of milk from the farm to the processing site and this is
due primarily to the growth of psychrotrophic bacteria, including pseudomonads. Critical sites
in the milk tanker for cleaning have been identified as the air separator, the milk meter, the milk
sieve, and the suction hose, and factors that contribute to inadequate cleaning include blockage
of the cleaning-in-place (CIP) spray system and low water pressure and flow rate. These can
lead to buildup of milk stone on the inner surface of the tanker.
Although Gram-negative psychrotrophic bacteria present in raw milk do not survive
pasteurization, these organisms are commonly isolated from pasteurized milk and cream, again
with Pseudomonas spp. being the most frequently encountered. Thus, the shelf life of
pasteurized products is limited by post-pasteurization contamination. A likely cause of postpasteurization contamination in the processing plant is shedding of bacteria from biofilms
formed on gaskets in pasteurized milk pipelines. The biofilm is generally defined as a complex
structural, heterogeneous, genetically divergent community of microorganisms that exist on a
solid surface in the form of an extracellular matrix composed of polymeric compounds
(exopolysaccharides and/or lipopeptides). The nature of the extracellular three-dimensional
matrix, the ratio of proliferation and interaction between cells within the biofilm is determined
by the available conditions for growth, the medium and substrate (Watnik & Kolter, 2000;
Constantin, 2009). Bacteria in biofilms (sessile form) are more resistant to chemical sanitizers
and the majority of antibiotics than are the same bacteria in suspension (planktonic form)
(Mosteller & Bishop, 1993), leaving viable bacteria to be dislodged into the milk product.
Gram-negative bacteria like Pseudomonas are largely recognized by their capability to produce
large amounts of exopolysaccharides, which contribute to adhesion and biofilm growth
(Drenkard & Ausubel, 2002). Pseudomonas biofilms can develop on the sides of gaskets,
despite operation of CIP systems and represent a long-lasting source of permanent product
contamination. There is substantial evidence that the filling operation has the greatest influence
on the potential shelf life of pasteurized milk.
0.2 Psychrotrophic bacteria
Psychrotrophic bacteria are defined as group of different bacterial species that are able to grow
at 7 °C or less regardless of their optimal temperature of growth (IDF Bulletins, 1976).
Psychrotrophic microorganisms are rod-shaped and forced aerobic and include the Gramnegative genera (Pseudomonas, Alcaligenes, Acchromobacter, Aeromonas, Serratia,
Chromobacterium, Flavobacterium), and the Gram positive genera (Bacillus, Clostridium,
Corynebacterium, Streptococcus, Lactobacillus, Microbacterium).
Psychrotrophic bacteria are ubiquitous in nature, primarily in water and soil, including
vegetation and a small number can also be present in the air. Ubiquitous distribution of these
bacteria indicates a remarkable degree of their physiological and genetic adaptability (Spiers et
al., 2000). Their optimal metabolic activity is expressed at temperatures between 20 and 30 °C.
However, they can grow and multiply at low temperatures through an enrichment of
polyunsaturated fatty acid in their membrane lipids. In other words, the altered cell membrane
secures sufficient permeability for membrane fluidity and transport activity of metabolites
necessary for growth and reproduction of bacteria at low temperatures (Schinik, 1999).
13
Preface
During cold storage after milk collection, psychrotrophic bacteria replaced Gram-positive
mesophilic aerobic bacteria and dominate the flora, leading to many quality problems in dairy
products (Cousin & Bramley, 1985; Lafarge et al., 2004; Ercolini et al., 2009). Under sanitary
conditions, <10% of the total microflora are psychrotrophs, compared to >75% under
unsanitary conditions (Suhren, 1989). The numbers of psychrotrophs that develop after milk
collection depend on the storage temperature and time.
Typically, 65–70% of the psychrotrophs isolated from raw milk are Pseudomonas species
(Griffiths et al., 1987; García et al., 1989). Other important associated-raw milk psychrotrophs
include members of the genera Bacillus, Micrococcus, Aerococcus, and Lactococcus and of the
family Enterobacteriaceae (genera Serratia) (Nörnberg et al., 2010).
Pseudomonads are Gram-negative, straight or curved rods, which are motile by polar flagella.
They are aerobic and their metabolism is never fermentative. They are catalase-positive and the
majority of species are oxidase-positive. Some of the species show distinguishable colony
morphologies or pigmentation (i.e., the blue-green derivative of phenazine, pyocyanin, and the
yellow-green fluorescing pigments; Kıska & Gilligan, 1999). Pseudomonas fluorescens is found
predominantly in soil and water and it produces a diffusible fluorescent pigment, pyoverdin.
The taxonomy of the genus Pseudomonas is complex and there is extensive genetic
heterogeneity among its members. In comparison to other psychrotrophic bacteria,
Pseudomonas spp. are characterised by a short generation time (<4 h) at 0-7 °C and the lowest
theoretical minimum growth temperature of -10 °C, which is close to that of typical
psychrophiles (Chandler & McMeekin, 1985; Sorhaug, 1992). Pseudomonas spp., with
predominance of P. fluorescens, are the most commonly isolated bacteria in raw and
pasteurized milk at the time of spoilage (Cousin, 1982; Sørhaug and Stepaniak, 1997). Spoilage
is occurred as the change of flavour, undesirable coagulation of milk proteins, and the increased
concentration of free fatty and amino acids. In addition, depending on the type of dairy product,
the atypical texture and proportion of certain undesirable organic compounds are occurred
(Cox, 1993; Boor & Murphy, 2002; McPhee & Griffiths, 2002; Cempírková & Mikulová,
2009). With regard to other quality aspects, such as suitability of milk for the production of
dairy products, psychrotophs have a significant negative effects on yields as well as on limiting
shelf life of dairy products (Cousin, 1982).
In addition to the ability to grow and multiply at low temperatures, psychrotophic bacteria have
the ability to produce heat-stable extracellular hydrolytic enzymes (Cousin, 1982; Chen et al.,
2003).
0.3 Enzymes in milk
0.3.1 Indigenous milk enzymes
Bovine milk is a biologically active product. Fifty to sixty different indigenous enzyme
activities have been reported in clean, freshly drawn milk (Andrews, 1991; Muir, 1996a). The
levels of these enzymes are not constant. Factors that influence this variability in enzyme levels
include the breed and age, stage of lactation, diet and nutrition, health status of the cow, season
(Andrews, 1991; Deeth & Fitz-Gerald, 1994). However, only a few of the indigenous milk
enzymes (i.e. enzymes in fresh unprocessed milk) have a substantial impact on the quality and
shelf life of milk and milk products (Muir,1996a). The most important of these are proteases
and lipases.
Two particular indigenous milk proteases have been studied in detail: milk alkaline proteinase
(MAP, now referred to as ‘‘plasmin’’, a serine protease; Reimerdes, 1981) and milk acidic
proteinase (cathepsin D, an aspartic protease; Kaminogawa & Yamauchi, 1972; Larsen et al.,
14
Preface
1996). These indigenous proteases arise from mammary tissue cells, blood plasma or leucocytes
(Andrews, 1991).
The most studied indigenous milk lipase was milk lipoprotein lipase (LPL), a dimer of
glycoprotein chains, each of 42 kDa, containing 8.3% carbohydrate. LPL is synthesized in
mammary gland secretory cells (Deeth & Fitz-Gerald, 1976).
Although indigenous milk enzymes may affect the shelf-life of milk, the accumulation and
action of extracellular proteolytic and lipolytic enzymes, produced by psychrotrophic bacteria,
are mainly correlated with quality deterioration in milk and dairy products.
0.3.2 Bacterial extracellular enzymes
Apart from indigenous enzymes, milk (raw or processed) also contains enzymes originating
from contaminating psychrotrophic bacteria, responsible for the highest spoilage of milk and
dairy products during storage.
In addition to being able to grow rapidly in refrigerated milk, psychrotrophs produce
extracellular enzymes (proteases, lipases and lecithinases) that can degrade milk components,
producing functional and flavor defects. Populations of psychrotrophs ranging from 10 6 to 107
cfu/ml-1 can produce sufficient amounts of extracellular enzymes to cause defects in milk that
are detectable by sensory tests (Fairbairn & Law, 1987). Extracellular enzyme production by
psychrotrophs is normally in the late exponential or early stationary phase of growth (Fox &
Stepaniak, 1983; Griffiths, 1989; Kohlmann et al., 1991). Optimal enzyme synthesis occurs in
majority of psychrotrophs at 20-30 °C, but considerable synthesis occurs at lower temperatures.
For example, production of extracellular protease by Pseudomonas fluorescens at 5 °C was
55% of that produced at 20 °C (Mckellar, 1982).
Although most psychrotrophs present in milk do not survive pasteurization (Varnam &
Sutherland, 1994; Muir, 1996a) or ultra high heat treatment (UHT, 135-150°C for 1-4 s)
regimes (Griffiths et al.,1981; Suhren, 1989), in many cases the extracellular enzymes that they
produce are unlikely to be destroyed by the heat processes and remain active in the final treated
product (Muir, 1996c). Both proteases and lipases produced by psychrotrophs, representative of
a number of genera, retained 60–70% of their activity after heating at 77 °C for 17 s and about
30–40% of their activity remained after UHT treatment at 140 °C for 5 s. For example,
proteases from Pseudomonas species isolated from raw milk retained 55–65% of the initial
activity after a heat treatment at 77 °C for 17 s and 20–40% activity after heat treatment at 140
°C for 5 s in buffers at pH 7 (Griffiths et al., 1981). The crude lipase produced by a
psychrotrophic Pseudomonas species retained 75–100% activity after heat treatment at 100°C
for 30 s in skim milk (Fitz-Gerald et al., 1982). Lipases are produced concomitantly with
proteases by the same bacterium and are generally more heat-stable than the proteases (Driessen
& Stadhouders, 1974; Griffiths et al., 1981; Fox & Stepaniak, 1983; Chen, 2000). Among the
features that stabilize heat-resistant enzymes from psychrotrophic microorganisms are
additional salt bridges and hydrogen bonds, tighter Ca 2+ binding sites, maximed packing,
shorter loops and an expanded hydrophobic core (Stepaniak & Sorhaug, 1995). Psychrotrophic
enzymes are not active (heat –labile) above 50-60 °C, in particular, the low temperature
instability of Pseudomonas protease was in the 50-60 °C range while the temperature of
minimum stability for lipases from Pseudomonas was 60-80 °C (McKellar, 1982; Stepaniak &
Sorhaug, 1995). The inactivation of most extracellular enzymes from psychrotrophs would thus
require heat treatments that are completely unacceptable in the dairy industry.
A variety of factors, including quorum sensing, growth phase, environmental and nutritional
factors (such as iron availability), are involved in the regulation of synthesis of extracellular
enzymes. For example, pH, temperature, oxygen tension, adenosine triphosphate pools,
presence of ions, organic nutrients, triglycerides, and many more have been found to influence
15
Preface
enzyme synthesis. In a Belgian study (Marchand et al., 2009), the incidence of proteolytic
psychrotrophs was lower in milks collected in winter than in summer but the strains isolated in
winter exhibited greater proteolytic activity than their summer counterparts. Surprisingly, the
regulation of synthesis of extracellular enzymes by Pseudomonas is not unequivocally
established, and probably involves a variety of regulatory mechanisms acting in concert. This
fact further highlights the complexity and diversity of Pseudomonas extracellular enzymes.
Understanding the complex mechanisms that direct enzyme synthesis will provide strategies to
target for control (McPhee & Griffiths, 2002).
An interesting consequence of enzyme activity from psychrotrophs is the stimulation of the
growth of starter lactic acid bacteria (LAB) in milk. Presumably this is because LAB can utilize
peptides, amino acids and ammonia that accumulate in milk and are produced by
psychrotrophic proteases. On the other hand, free fatty acids releases by lipases may inhibit the
growth of LAB (Sørhaug & Stepaniak, 1991; Jaspe et al., 1995).
0.3.2.1 Proteases
In general, the proteases of psychrotrophic bacteria degrade milk casein (producing different
peptides with altered compositional, structural and physical characteristics), leading to the
formation of a gel structure or coagulation of sterilized milk during storage (Harwalker et al.,
1993; Datta & Deeth, 2003) Development of astringency in some raw milk and in pasteurised or
ultra-high temperature (UHT)-sterilised milk samples during storage has been related to the
production of polypeptides by proteases that survive UHT treatment (Sørhaug & Stepaniak,
1997). In cheese making, proteases (which are not extracted by whey) cause a significant yield
loss (Cousin, 1982; Mitchell & Marshall, 1989). Furthermore, proteolysis caused by
psychrotrophic bacteria has a negative effect on the products flavour, which has been described
as bitter, foreign, unclean, fruity, yeasty or metallic (Marshall, 1982). Most of the proteases
isolated from Pseudomonas are metalloenzymes with molecular mass between 40-50 kDa,
containing at least 1 zinc atom and up to 16 calcium atoms per molecule. They are rich in
alanine and glycine residues and poor in cysteine and methionine residues and calcium is
essential for the activity and stability of these proteases (Mitchell et al., 1986).The optimum pH
of the Pseudomonas proteases are neutral (~7) or alkaline (7-9) and temperature optima range
from 30-45 °C; in all cases, activity decreases sharply at temperatures above the optimum but
the proteases retain partially activity also at lower temperatures (4 °C) (Mitchell & Ewings,
1985).
In milk, Pseudomonas proteases preferentially hydrolyze κ-casein > β-casein > αS1-casein. The
proteins of the lactoserum are very few hydrolyzed (Cousin, 1982; Fairbairn & Law, 1986;
Koka & Weimer, 2000; Rajmohan et al., 2002). One extracellular metalloprotease called AprX,
which belongs to the serralysin family, has been characterized in P. fluorescens and its gene
was identified in several strains (Martins et al., 2005).
0.3.2.2 Lipases and Phospholipases
Psychrotrophic lipases, catalysing the hydrolysis of triacylglycerols, lead to the accumulation of
free (non-esterified) fatty acids, partial glycerol esters (monoacylglycerols, diacylglycerols) and
even glycerol in some cases in milk (Deeth, 2006). The products of lipolysis are highly
detrimental to the formation and stability of milk foams that are colloidal systems in which air
bubbles are stabilized by a matrix composed of milk components. The depression of milk
foaming, caused by lipolysis, is due to the partial glycerides, which are surface active and
displace the foam-stabilizing proteins at the air-water interface of the foam bubbles. Besides,
FFAs are the primary cause for the changes in product flavour that is described as rancid,
16
Preface
unclean, soapy, butyric, astringent or bitter, which could influence consumer acceptance of milk
and dairy products. The lipolytic flavours defects are particularly pronounced in cream, butter,
cheese and sterilised (UHT) milk (Stead, 1986; Champagne et al., 1994).
In general, psychrotrophic lipases have molecular masses ranging from 30 to 50 kDa, pH
optima between 7 and 9 and temperature optima range from 22 to 55 °C. From the patterns of
hydrolysis of triacylglycerols bacterial lipases are divided into two major groups, I and II.
Group I (non-specific) lipases catalyse the production of glycerol by releasing fatty acids from
all the three positions; group II (1,3-specific) lipases catalyse the formation of 1,2- and 2,3diacylglycerols and 2-monoacylglycerols with the release of fatty acids from positions 1 and 3
of the triglyceride. In the case of group II lipase products, acyl migration leads to the production
of 1,3-diacylglycerols and 1-monoacylglycerol and subsequently the production of glycerol
over an extended period (Macrae, 1983).
Lecithinases are important groups of phospholipases of psychrotrophic bacteria that are able to
disrupt the protective membrane structure of fat globules and milk fat become available to the
native milk lipases resulting in physical degradation of the emulsion in milk (Shah, 1994).
The ability of psychrotrophic bacteria to produce lipases and phospholipases varies
considerably across genera, as well as among species of the same genera (Witter, 1969).
0.4 Control of psychrotrophs and related enzymes
The negative impacts of psychrotrophic bacteria on the quality of milk and dairy products are
unquestionable. Their ubiquitous nature in the production environment , the ability for rapid
growth under low temperatures and the capacity to synthesize thermo-stable extracellular
enzymes, have made this group of bacteria the leading cause of spoilage of milk and dairy
products. Contamination of raw milk with psychrotrophs, even under the best manufacturing
practices, cannot be completely avoided. However, cooling of milk at a temperature of 2 °C,
instead of at temperatures between 4 and 6 °C (which is the most commonly applied cooling
temperature), can significantly slow their growth as well as their proteolytic and lipolytic
activity, only in cases where their initial number is ≤103 cfu mL-1 (Kumarsan et al., 2007). The
heat treatment of raw milk (65-69 °C/15 s) in the dairy plant prior to pasteurization can reduce
the number of Gram-negative psychrotrophic bacteria by 77-97 % (Champagne et al., 1994).
Other examples of methods currently proposed to control psychrotrophic bacteria and/or their
extracellular enzymes include additives (CO2, nitrogen), high-pressure treatment, modified
atmosphere storage of raw milk, microbial antagonism, activation of the lactoperoxidase system
in milk, addition of enzyme inhibitors, addition of bacteriocin-producing lactic acid bacteria,
and low temperature inactivation of enzymes, to name a few (Champagne et al., 1993; Shah,
1994; Sørhaug & Stepaniak, 1997).
However, adequate cleaning and permanent hygiene control in all aspects of milk handling,
transport and processing, strict maintenance of refrigeration at 4 °C or lower, minimization of
the storage time of raw milk, and a suitable method to kill or remove psychrotrophic bacteria,
followed up by an effective HACCP system, are all primary concerns for quality assurance in
the dairy industry. Most practices currently employed in the industry focus on elimination of the
bacteria by heat processing but it is clear that bacterial numbers (i.e. viable bacteria) do not
correlate with enzyme levels since the enzymes survive treatments that kill the bacteria, and
cannot give an accurate indication of product quality. Therefore it would be useful to develop a
quick and simple enzyme assay method that can provide a direct correlation between enzyme
activities and levels and keeping quality of dairy products. The dairy industry would be able to
concentrate its efforts on control of the most problematical bacteria (and consequently control
of the levels of enzyme introduced into products) and therefore maximize processing efficiency.
17
Preface
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22
State of the art
1. STATE OF THE ART
Milk is a rich growth medium for microorganisms, which can increase rapidly during storage
thus affecting the technological properties of raw milk. Refrigeration has an important impact
on the microbiological quality of raw milk by decreasing the presence of mesophilic bacteria.
However, cold storage of unprocessed milk creates selective conditions for the development of
psychrotrophic bacteria, that are able to grow below 7 °C (Champagne et al., 1994). The
psychrotrophs from refrigerated milk include both Gram-negative and Gram-positive bacteria,
belonging to numerous genera. Pseudomonas spp. dominate the psychrotrophic microflora of
raw or pasteurized milk at the time of spoilage (Sørhaug & Stepaniak, 1997); for this reason up
to now most of the research has been focused on Pseudomonas genus, which includes the
predominant microorganisms affecting the shelf-life of processed fluid milk at 4 °C. P.
fluorescens, P. putida, P. fragi, P. putrefaciens, are the most isolated species and less frequently
P. aeruginosa (Gilmour & Rowe, 1990). Significant contaminations by psychrotrophs occur
due to inadequately sanitized surfaces of milking, storage and transporting equipments.
Furthermore post-pasteurization contaminations may happen at the filling operation (Mac Phee
& Griffiths, 2002).
Psychrotrophs are responsible for the highest spoilage of milk because of their capacity to
produce thermostable extracellular enzymes (proteases, lipases and lecithinases), which have
major effects on the quality of raw milk and milk products (Sørhaug & Stepaniak, 1997).
Although most psychrotrophic bacteria are destroyed by the conventional thermal treatments
(pasteurization and UHT processing) employed in the dairy industry, such treatments have
minor effects on their enzymes; they can resist and continue to degrade milk in the absence of
viable bacterial cells, reducing the shelf-life of milk and dairy products (Cousin, 1982; Koka &
Weimer, 2000; Chen et al., 2003).
Enzymes can also be detrimental to the quality of cheese by causing bitter or rancid flavors and
by impairing the coagulation properties of the milk (Richter & Vedamuthu, 2001). Proteases
degrade casein, resulting in bitterness in milk, gelation of UHT-sterilized milk and decreased
yields of soft cheese. Lipases by hydrolyzing triglycerides to free fatty acids and glycerol,
produce flavor defects (rancid, butyric, astringent, soapy) in cream, butter, cheese and UHT
products. Pseudomonas spp. are the primary concern with regard to lipolytic degradation of
milk fat (MacPhee & Griffiths, 2002). Lecithinases are able to disrupt the integrity of milk fat
globule membrane (MFGM), increasing the susceptibility of milk fat to the action of lipases
(Herrera, 2001; Ray, 2004).
Most of the research has been focused on Pseudomonas spp. (Eneroth et al., 1998; Martins et
al., 2006), but there is growing evidence that other genera, such as Enterobacteriaceae, may
have similar relevance by producing strong lipo-proteolytic activity (Nörnberg et al., 2010).
The enzymes are generally good indicator of the keeping quality of protein and lipid-rich foods
and the knowledge of their impact and deleterious effects on products may lead to the definition
of pertinent indicators and possible markers that can be used for quality control during
processing (Cousin et al., 2001).
Over the past years, although many studies have assessed the microbial diversity in raw milk,
just a few have provided more information on the development of off-flavors, on the release and
levels of FFAs from milk fat and on identification of peptides, related to the lipo-proteolytic
activity of psychrotrophs, that can be directly linked to functional and sensory properties and
quality of milk.
Moreover, scant information about other specific psychrotrophic microorganisms and the
effects of their enzymes on milk quality is available.
23
State of the art
1.1 References
Champagne CP et al., 1994, Psychrotrophs in dairy products: their effect and their control. Crit
Rev Food Sci Nutr 34:1-30.
Chen L et al., 2003, Detection and impact of protease and lipase activities in milk and milk
powders. Int Dairy J 13:255–275.
Cousin MA 1982, Presence and activity of psychrotrophic microrganisms in milk and dairy
products: a review. J Food Prot 45:172–207.
Cousin MA et al. Psychrotrophic microorganisms. In Compendium of methods for the
microbiological examination of foods, Downes FP, Ito K, eds., American Public Health
Association, Washington, DC. 2001, pp 159–166.
Eneroth A et al., 1998, Critical contamination sites in the production line of pasteurised milk,
with reference to the psychrotrophic spoilage flora. Int Dairy J 8:829–834.
Gilmour A, Rowe MT. Microorganisms associated with milk. In The Microbiology of Milk
Vol.1, 2nd, Robinson RK, ed., Dairy Microbiology, Elsevier Applied Science, London 1990,
pp. 37-75.
Herrera AG. Psychrotrophic microorganisms. In Food Microbiology Protocols. Spencer JFT,
Spencer de ALR, eds., Humana Press Inc., Totowa, New Jersey, NJ 2001, pp 3–11.
Koka R,Weimer BC, 2000, Isolation and characterization of a protease from Pseudomonas
fluorescens RO98. J Appl Microbiol 89:280–288.
Martins ML et al., 2006, Genetic diversity of Gram-negative, proteolytic, psychrotropic bacteria
isolated from refrigerated raw milk. Int J Food Microbiol 111:144–148.
McPhee JD, Griffiths MW. Pseudomonas spp. In Encyclopedia of Dairy Sciences, Vol. 4,
Roginski H, Fuquay WJ, Fox FP, eds., Academic Press 2002 , pp. 2340-2350.
Nörnberg MFBL et al., 2010, Proteolytic activity among psychrotrophic bacteria isolated from
refrigerated raw milk. International J Dairy Technol 63:41–46.
Ray B. 2004, Fundamental Food Microbiology. CRC Press, Boca Raton, FL.
Richter R., Vedamuthu ER, Milk and Milk Products. In Compendium of Methods for the
Microbiological Examination of Foods, Downes FP, Ito K, eds., American Public Health
Association, Washington, DC 2001, pp. 483–495.
Sørhaug T Stepaniak L,1997, Psychrotrophs and their enzymes in milk and dairy products:
Quality aspects. Food Sci Technol 8:35-41.
24
Aims of the study
2. AIMS OF THE STUDY
Hygiene in all aspects of milk handling, reduction of storage times, use of effective refrigeration
temperatures, availability of methods for reduction of pathogenic and spoilage microbial flora,
and an effective employment of HACCP systems are principal issues for the dairy industry.
In recent years, also in Lombardy, frequent technological and sensory troubles arose from
processing of milk containing psychrotrophic bacteria, responsible for the highest spoilage of
raw or heated milk during storage. Indeed, nowadays, these bacteria represent a constant and
major cause of concern for the dairy industry with regard to their capacity to synthesize
thermostable extracellular enzymes. Such enzymes can hydrolyse milk fats and proteins leading
to development of gelation, off-odours/flavours, loss of sensory quality and shelf life. For these
reasons, this research project aims to acquire further knowledge on the enzymatic traits of raw
milk-associated psychrotrophic bacteria. On these bases, the research also deals with the
detection of specific molecules and metabolites derived from by bacterial enzymatic
degradation of milk components. In particular, in this study, we attempted to assess the
hydrolytic activities of 80 psychrotrophic bacteria, belonging to different genera, isolated from
raw bovine milk samples collected at different farms located in Lombardy. Moreover, we
evaluated their spoilage potential by analyzing the production of VOCs and the release of FFAs
in milk and then we characterize their extracellular proteolytic activity by means the evaluation
of caseinolytic potential, identification of secreted proteases and characterization of peptide
profile generated by the action of bacterial protease.
Overall, the final aim of this PhD thesis was to recognize particular molecules and metabolites
derived from bacterial enzymatic degradation of milk components useful as a good markers to
early recognize the activity for psychrotrophic strains in raw milk.
25
Results and discussion: Characterization of Gram-negative psychrotrophic bacteria
3. RESULTS AND DISCUSSION
3.1 Characterization of Gram-negative psychrotrophic bacteria isolated from raw milk
The combination of longer storage times and lower temperatures of raw milk throughout the
dairy chain prior to heat treatment creates selective conditions for growth of thermo-resistant
enzyme-producing psychrotrophic bacteria, altering the structural and sensory properties of the
finished dairy product (Chen et al., 2003).
Psychrotrophs are bacteria that can grow at temperatures between 0°C and 7°C, although their
optimal growth temperature is higher (Champagne et al., 1994; Sørhaug & Stepaniak, 1997) and
can constitute up to 70–90% of the microbial population in raw milk stored at low temperatures
(Cousin, 1982; Sørhaug & Stepaniak, 1997). Psychrotrophic bacteria from numerous genera
have been isolated from milk, the genus Pseudomonas generally dominating the microbiota of
refrigerated raw milk, (Eneroth et al., 2000; Ercolini et al., 2009; Rasolofo et al., 2010; De
Jonghe et al., 2011; Raats et al., 2011;), but also reported to be prevalent are the
Enterobacteriaceae (Nörnberg et al., 2009). Psychrotrophic pseudomonads are recognized as
major spoilage microorganisms of milk and dairy products due to the production of heat-stable
extracellular enzyme, proteases, lipases and lecithinases (Cousin, 2001; Herrera, 2001).
Although psychrotrophic bacteria are easily destroyed by the heating settings applied by the
dairy industry, their enzymes can survive and remain active in processed milk and derived dairy
products (Deeth, 2002; Ray, 2004; Bhunia, 2008). Proteases are predominantly active towards
the casein fraction, producing a grey colour and bitter off-flavours in milk, gelation of UHT
sterilized milk and decreased yields of soft cheese. Most proteases are metalloproteases, which
are rich in alanine and glycine residues and poor in cysteine and methionine residues; calcium is
essential for the activity and stability of these proteases (Mitchell et al., 1986). In milk, they
preferentially hydrolyze casein κ, then casein β, then casein αS1.
The aprX gene, which encodes a heat-resistant alkaline metalloprotease, belongs to the
serralysin family, and has been characterized in several strains of Pseudomonas spp. (Duong et
al., 1992; Liao & McCallus, 1998; Kawai et al., 1999; Kumeta et al., 1999; Chessa et al., 2000;
Chabeaud et al., 2001), and extensively studied only in P. aeruginosa and P. fluorescens. AprX
is believed to be responsible for the spoilage of milk, and has been proposed as a marker for
milk bacterial contamination, with high protein degrading effects (Dufour et al., 2008).
Lipases catalyze the hydrolysis of triglycerides, which leads to rancid, butyric, astringent, soapy
flavours and the depression of milk foaming properties (De Jonghe et al., 2011). Lipolytic
degradation of milk is not as predominant as proteolytic degradation, but the free fatty acids
released from milk fat hydrolysis, caused by psychrotrophic lipase activity, are the primary
cause of changes in product flavor, described as rancid, unclean, soapy or bitter, making the
product barely acceptable (Ma et al., 2000; Deeth, 2006).
Psychrotrophic bacteria are particularly troublesome in milk, also for their lecithinase enzymes
that act on the phospholipids of milk fat globule membranes (MFG); the disruption of the MFG
results in an unstable fat emulsion (flocks on the milk and cream surfaces) and the
triacylglycerols are then easy targets for bacterial lipases (Shah, 1994; Herrera, 2001; Fox,
2002, Ray, 2004).
In recent years in Italy, there have been frequent technological and sensory troubles caused by
both the proteolytic and the lipolytic activity of psychrotrophic bacteria in UHT milk and also
in pasteurized milk.
Due to the relevance of the aforementioned issues, and as the psychrotrophic microbiota is the
most important in the process of enzymatic milk spoilage, a more detailed evaluation on
proteolytic, lipolytic and lecithinase activities is crucial for the development of control
strategies, increasing both the sensory quality and the shelf life of milk.
26
Results and discussion: Characterization of Gram-negative psychrotrophic bacteria
Thus the first study of this PhD thesis aimed to (i) isolate and identify Gram-negative
psychrotrophic bacterial strains from unprocessed bulk milk collected from different farms
located in Lombardy; (ii) assess the bacterial lipolytic, proteolytic and lecithinase traits that
could influence the milk itself, and the dairy product shelf life in different conditions; iii)
evaluate the presence of aprX gene in psychrotrophic strains.
3.2 Material and methods
3.2.1 Psychrotrophic bacterial counts and isolation of gram-negative psychrotrophic
(GNP) strains
Eighty psychrotrophic strains were isolated from 56 summer and winter samples of unprocessed
bulk milk collected from 28 different farms in the Lombardy region of Northern Italy; the total
production was destined to produce Grana Padano P.D.O. cheese. Bulk milk was refrigerated at
11°C after the two daily milkings, following the indications of Grana Padano P.D.O. production
rules. At each farm, raw milk was sampled twice over a 6 month period, in January and July.
The milk samples were transported to the laboratory in sterile boxes, stored at 4°C, and
analyzed within 24h. Decimal progressive dilutions were carried out with quarter-strength
Ringer's solution (Scharlau, Barcelona, Spain). Psychrotrophic bacteria were isolated on Plate
Count Agar (Oxoid, Ltd., Basingstoke, UK) at 7°C after 10 days of incubation. Two colonies
with different and distinguishable morphologies were picked out from each sample, and
subcultured on Brain Heart Infusion (BHI) Agar (Biolife, Milan, Italy) to obtain pure cultures.
The presumptive Pseudomonas colonies were preliminarily confirmed either on Pseudomonas
Selective Agar (Biolife), after incubation at 37°C during 18-42 h, and on Penicillin-Pimaricin
(Biolife) supplemented Pseudomonas agar base (Oxoid) plates according to ISO-13720:2000,
incubated at 25°C for 48 h. The isolates were maintained and propagated in BHI broth (Biolife)
and incubated under aerobic conditions at 30°C overnight. The strains were stored at -20°C in
litmus milk until use.
3.2.2 Phenotypic characterization and biochemical tests
Pure cultures were Gram-stained and examined for growth at different temperatures, motility,
pigment production, oxidase and catalase activity, gas and extracellular enzymes production.
The bacterial isolates were incubated in Brain Heart Infusion broth (Biolife) at 7, 22 and 30 °C
until growth occurred. Motility was measured as the diameter of zone travelled by bacteria
point-inoculated into Soft-Luria-Bertani (SOFT-LB) agar plates (10 g L-1 tryptone, 5 g L-1 yeast
extract , 10 g L-1 NaCl, 3 g L-1 agar ). Plates were incubated 18–20 h at 30 °C and halo diameter
measurements were recorded. Bacterial motility was negative for halo diameter < 0,5 cm. The
colony pigmentation was visualized on “Mascarpone agar” plates (1 kg L-1 mascarpone cheese
30 g L-1 agar, 10 g L-1 yeast extract) incubated at 30 °C for 24 h to 72 h. The oxidase reaction
was determined using BBLDrySlide oxidase slides (Becton Dickinson Company, Sparks, Md.
USA), according to the manufacturer’s instructions. For catalase activity, 1.5% H2O2 was
dropped onto a bacterial smear that had been placed on a glass slide. Gas production indicated a
positive reaction. All isolates were checked for gas production from glucose in BHI broth
(Biolife) containing Durham tubes.
All the strains were tested for their proteolytic, lipolytic and lecithinase activities by agar
diffusion method assay at 7, 22 and 30 °C up to 20 days (Tables 3.3.3 and 3.3.4). Proteolytic
enzyme production was visualized on skim milk agar (5% skim milk powder, 3% agar). After
incubation, the presence of a clear zone around the colonies was indicative of proteolysis.
Lipolytic strains were screened using Tributyrin Agar plates (Oxoid), and the activity of the
27
Results and discussion: Characterization of Gram-negative psychrotrophic bacteria
lipase was observed as a zone of hydrolysis around the bacterial colonies (Meghwanshi et al.,
2006). The production of extracellular phospholipases (lecithinases) was determined on Plate
Count Agar (Oxoid) supplemented with 10% egg yolk emulsion (Biolife), as described by
Dogan and Boor (2003). Colonies surrounded by an opaque ring were deemed to evince
lecithinase activity (Bates & Liu, 1963).
3.2.3 DNA extraction
DNA extraction was carried out using 1 ml of a BHI broth overnight culture containing
approximately 1 -3 * 108 cells. DNA was extracted using the Microlysis kit (Labogen, Rho,
Italy) following the manufacturer’s instructions.
3.2.4 Identification of psychrotrophic isolates by 16S rRNA and rpoB genes sequencing
The 16S rRNA gene was amplified by polymerase chain reaction (PCR) using the universal
primers
p8FLP
(5’-AGTTTGATCCTGGCTCAG-3’)/p806R
(5’
GGACTACCAGGGTATCTAAT-3’) (Mc Cabe et al., 1995) to generate an amplicon of ca. 800
bp. PCR amplification was carried out in a Mastercycler (Eppendorf, Hamburg, Germany) with
the following conditions: initial denaturation at 94 °C for 5 min followed by 30 cycles of 94 °C
for 1 min, 56 °C for 1 min and 72 °C for 1 min. Final extension was carried out at 72 °C for 5
min. Each DNA amplification was performed in 200 µl microtubes using a 25 µl reaction
mixture containing 50–100 ng DNA template, PCR Master Mix 2X (Fermentas, Inc.,
Burlington, Ontario, Canada), 10 µM of the primer pair and double-distilled water to achieve
the final volume.
Since the 16S rRNA gene is known for its low resolution within Pseudomonas at the species or
subspecies level (Yamamoto et al., 2000; Ercolini et al., 2007), partial sequence analysis of the
rpoB gene was also performed. PCR amplification of rpoB was performed with the set of
primers PSF (5'-AGT-TCA-TGG-ACC-AGA-ACA-ACC-3') as forward/ rpoB-PTR (5'-CCTTGA-CGG-TGA-ACT-CGT-TTC-3') as reverse under the conditions described by Sajben et al.
(2011). The amplification started with an initial denaturation at 94°C for 3 min, followed by 30
cycles of denaturation at 94°C for 1 min, annealing at 58°C for 1 min, and elongation at 72°C
for 1 min, and a final extension step at 72°C for 10 min. The PCR-amplified 16S rRNA and
rpoB gene products were separated on a 1.5% agarose gel (GellyPhor, Euroclone) stained with
SYBR Safe (Invitrogen, Minneapolis, MN, USA) and photographed using a UV
transilluminator. Molecular size markers (100-bp DNA ladder, Euroclone) were included in
each agarose gel.
Amplification products were sent to Macrogen Europe (Amsterdam, Netherlands) for
sequencing. Concerning data analysis, partial rpoB and complete 16S rRNA gene sequences
(approximately 310bp and 800bp, respectively) were analyzed with NCBI BLAST search
(http://www.ncbi.nlm.nih.gov/BLAST, Altschul et al., 1990). Species names were assigned
whenever the degree of homology was higher than 97%.
3.2.5 Randomly amplified polymorphic DNA (RAPD) analysis
Gram-negative psychrotrophic bacteria isolated from unprocessed milk samples were RAPDtyped to study their genetic variability and to distinguish closely related strains. RAPD-PCR
reactions were performed with primers M13 (5′-GAGGGTGGCGGTTCT-3′) (Huey & Hall,
1989) and OPAA-10 (TGGTCGGGTG) (Martins et al., 2006). Briefly, the amplification with
M13 started with an initial denaturation at 94°C for 2 min, followed by 35 cycles of
denaturation at 94°C for 1 min, annealing at 45°C for 20 sec, and elongation at 72°C at
28
Results and discussion: Characterization of Gram-negative psychrotrophic bacteria
0,5°C/sec, and a final extension step at 72°C for 2 min (Andrighetto et al., 1998).While the
amplification with OPAA-10 was carried out using the following protocol: 40 cycles consisting
of denaturation at 94°C for 1 min, primer annealing at 37°C for 1 min, and extension at 72°C
for 90 s. To complete the synthesis of all strands, the procedure was concluded with extension
at 72°C for 7 min (Martins et al., 2006). Grouping of the RAPD-PCR profiles was obtained
with the BioNumeric 5.1 software package (Applied Maths, Kortrjik, Belgium) using the
UPGMA (unweighted pair group method using arithmetic averages) cluster analysis. The
reproducibility value of the RAPD-PCR assay, calculated from two repetitions of independent
amplification of psychrotrophic type strains, was higher than 90%.
3.2.6 Detection of the aprX gene in psychrotrophic strains (aprX –PCR)
The presence of the aprX gene, encoding for a known alkaline heat-resistant metalloprotease,
was searched for in psychrotrophic bacteria, using an aprX-PCR test. PCR amplification of the
aprX gene was performed with the set of primers SM2F (5'-AAA-TCG-ATA-GCT-TCA-GCCAT-3')/SM3R (5'-TTG-AGG-TTG-ATC-TTC-TGG-TT-3') with an amplification product of
approximately 850 bp, under the conditions described by Marchand et al. (2009). PCR
amplification was carried out in a Mastercycler (Eppendorf), and the cycle parameters were 5
min at 95°C for initial denaturation followed by 30 cycles with denaturation for 30 s at 95°C,
annealing for 30 s at 60°C, extension for 1 min at 72°C and a final elongation step of 72°C for 8
min.
3.3 Results and discussion
3.3.1 Psychrotrophic bacterial counts and isolation of gram-negative psychrotrophic
(GNP) strains
The psychrotrophic bacterial content in the 56 raw milk samples analyzed in this study ranged
between 2.43 and 6.46 log cfu ml-1, representing from 49.1 to 99.99 % of Standard Plate Count
(SPC). Total count of mesophilic bacteria in the whole set of bulk milk samples ranged from
3.78 to 6.89 log cfu ml-1. SPC values are higher than those previously reported in Italy (Bava et
al., 2011) as a consequence of the refrigeration temperature (> 8 °C) required by Grana Padano
production rules. Eighteen milk samples contained more than 5 log-units total bacterial count,
being the average content of psycrotrophs 91,63% ± 14,40 which not differed from that of milk
samples characterized by lower bacterial count (93,63 % ± 13,33).
Psychrotrophs make a contribution to SPC similar to that reported by other authors in Brasil
(Nörnberg et al., 2010) and Italy (Bava et al., 2011); differently, Cempírková (2002), in a study
conducted on seven farms over an 18 month period, highlighted a high correlation (p<0.001)
between psychrotrophic Gram-negative bacteria (PB) and the total count of mesophilic aerobic
bacteria (TB), with a lower proportional PB/TB index (0.18). In the microbiological quality
assessment of raw milk in Denmark, Holm and others (2004) confirmed that psychrotrophic
bacteria were dominant in 28 % of the cases where TB exceeded 30,000 cfu mL-1. Contrary to
this, under conditions of unhygienic milking, the number of psychrotrophic Gram-negative
bacteria in raw milk accounted for 75-99 % of the total microbial population, with the
predominance of the P. fluorescens (Marshall, 1982; Cox, 1993; Muir, 1996).
The combination of longer storage times and lower temperatures of raw milk throughout the
dairy chain prior to heat treatment creates selective conditions for growth of thermo-resistant
enzyme-producing psychrotrophic bacteria, altering the structural and sensory properties of the
finished dairy product (Chen et al., 2003). In 1980, Gehringer identified that high raw milk
quality can only be achieved if the occurrence of psychrotrophic bacteria is less than 10 % of
29
Results and discussion: Characterization of Gram-negative psychrotrophic bacteria
the total bacterial count. However, cooling and holding refrigerated raw milk longer prior to
processing causes changes in the microbial population. Under such conditions, the dominant
Gram positive bacteria are replaced by Gram-negative and Gram-positive psychrotrophic
bacteria.
A total of 99 colonies were isolated from 56 different milk samples originating from 28 separate
dairy farms, in order to minimize the chances of them being the same strain even if the choice
to isolate 2 colonies with different morphologies from each sample do not allow to draw
conclusions about the prevalence of the species.
Of these, 80 were Gram-negative bacteria (80.8%), confirming the predominance of GNP in
milk, which has already been reported in other studies (Jayarao & Wang, 1999), and were
selected for further analysis. In this work bacterial isolates were identified at the species level
by partial rpoB gene sequencing in the case of low 16S rRNA gene sequence heterogeneity. In
particular the housekeeping rpoB gene, encoding the beta-subunit of RNA polymerase,
provided improved phylogenetic resolution over the 16S rRNA gene for the Pseudomonas
genus and can be used to complement the information gathered from the 16S rRNA gene, as
reported by previous studies (Moore et al., 1996; Anzai et al., 2000; Yamamoto et al., 2000;
Adékambi et al., 2009).
In total, 46 isolates were identified at the species level, based on 16S rRNA gene sequence, and
34 based on partial rpoB gene sequence since the sequencing of the 16S rRNA gene did not
enable species identification. Eight strains could not be associated with any previously
associated species, not even with the rpoB gene sequencing. Partial rpoB gene sequence
analysis recognized them as Pseudomonas sp., sharing an identity by more than 97%.
Among the identified GNP, Pseudomonas was the predominant genus with a prevalence of
78.75 %, followed by Enterobacteriaceae (21,25%). These findings are in agreement with
results of other researchers who stated that Pseudomonas represents a relevant part of raw milk
microbiota (Ercolini et al., 2009; Giannino et al., 2009) and that the total psychrotrophic raw
milk microbiota can comprise up to 90% Pseudomonas spp. (Champagne et al., 1994; Dogan &
Boor, 2003). On the contrary, some studies reported Enterobacteriaceae to be predominant
psychrotrophs in raw milk in Brasil and Spain (Suarez & Ferreiros, 1991; Nörnberg et al.,
2010). Enterobacteriaceae are frequently encountered in milk and during cheese production
(Sorhaug & Stepaniak, 1997; Morales et al., 2003; Mounier et al., 2005; Chaves-Lopez et al.,
2006), although they have been considered negative flora (eg. markers of hygiene, cheese
texture defects, blowing and off-flavours) and can be associated with raw milk samples from
mastitic animals (Nam et al., 2010). Among the Pseudomonadaceae, ten different species were
identified (Table 3.3.1). P. fluorescens and P. aeruginosa were the predominant species with a
percentage of 30.16 % and 22.22 % respectively, followed by P. putida (11.11 %), P. fulva
(6.34%), P. fragi and P. mosselii (4.76%), P. rhodesiae (3.17%). One strain of P. libanensis, P.
teatrolens and P. chlororaphis subsp. aurantiaca were found in raw milk samples. Among the
Enterobacteriaceae, Serratia marcescens was the most frequently isolated species, with a
percentage of 52.94 %, followed by Hafnia alvei (29.41%), Citrobacter freundii (11.76%) and
one strain of Enterobacter cloacae (Table 3.3.2). S. marcescens is the most frequently occurring
species of the Serratia genus and is found in soil, water, and sometimes on starchy foods such
as bread (Grimont & Grimont, 2006). Serratia strains have already been described in milk
(Tornadijo et al., 1993) and cheese (Martin-Platero et al., 2009) and have been shown to affect
milk and cheese sensory quality due to high proteolytic activity and dimethyl sulphide
production (Morales et al., 2003; Chaves-Lopez et al., 2006). In recent years, S. marcescens
has also been studied for its ability to produce lipase, which is responsible for flavour defects in
milk and dairy products during cold storage (Abdou, 2003). H. alvei has been found in raw milk
and raw milk cheeses (Tornadijo et al., 2001; Morales et al., 2005b; Kagkli et al., 2007), and
30
Results and discussion: Characterization of Gram-negative psychrotrophic bacteria
water was recently suggested as a possible contamination source for these species in milk
(Kagkli et al., 2007).
All the psychrotrophic strains considered in this study were characterised by RAPD-PCR
analysing amplification profiles obtained with two primers. A notable genotypic heterogeneity
and a high degree of variability among Pseudomonas and Enterobacteriaceae strains was
evident indeed a degree of homology higher than 90% was detected among strains belonging to
the same species only for 2 P. fluorescens (PS16 and PS24 strains) (Figure 3.3.1).
31
Results and discussion: Characterization of Gram-negative psychrotrophic bacteria
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
% Homology
PS17
Ps. putida
PS59
Ps. aeruginosa
PS68
Ps. putida
PR5
Ps. fluorescens
PS55
Ps. fragi
PS80
Ps viridiflava
PS45
Se. marcescens
PS90
Se. marcescens
PS69
Se. marcescens
PS92
Se. marcescens
PS36
Se. marcescens
PS74
Se. marcescens
PS91
Se. marcescens
PS30
Se. marcescens
PS33
Se. marcescens
PS40
Ps. putida
PS62
Ps. rhodesiae
PS65
Ps. aeruginosa
PS31
Ps. mosselii
PS57
Ha. alvei
PS75
PS54
Ps. putida
PS72
Ps. aeruginosa
PS61
Ps. aeruginosa
PS20
Ps. aeruginosa
PS25
En. cloacae
PS13
Ps. aeruginosa
PS31b
Ps. mosselii
PS39
Ps. mosselii
PS70
Ps. mosselii
PS88
Ps. aeruginosa
PS11
Ps. teatrolens
PS78
Ps. fluorescens
PS58
Ha. alvei
PS1
Ps. fulva
PS10
Ps. fragi
PS18
Ci. freundii
PS19
Ps. fluorescens
PS16
Ps. fluorescens
PS24
Ps. fluorescens
PS23
Ps. fluorescens
M3
Ps. fluorescens
M7
Ps. fluorescens
PS28
Ps. fluorescens
PS81
Ps. fluorescens
PS73
Ps. fluorescens
PS43
Ha. alvei
PS93
Ps. aeruginosa
PS6
Ps. chlororaphis subsp. aurant.
PS8
Ps. putida
PS14
Ps. fluorescens
PS49
Ps. libanensis
PS60
Ps. fluorescens
PS34
Ps. aeruginosa
M37
Ps. fluorescens
M44
Ps. fluorescens
PS42
Ps. aeruginosa
PS51
Ps. fulva
PS44
Ps. aeruginosa
PS53
Ps. aeruginosa
PS26
Ps. aeruginosa
PS77
Ps. fluorescens
PS32
Ps. aeruginosa
PS41
Ps. putida
PS85
Ha. alvei
PS12
Ps. fragi
PS38
Ps. aeruginosa
PR3
Ps. fluorescens
PS4
Ps. putida
PS66
Ps. aeruginosa
PS47
Ps. fragi
PS84
Pseudomonas spp.
PS29
Ps. aeruginosa
PS87
Ps. aeruginosa
PS67
Ps. aeruginosa
PS46
Ha. alvei
PS52
Ps. fulva
PS83
Ps. putida
PS56
Ps. fluorescens
PS86
Ps. aeruginosa
PS27
Ps. aeruginosa
PS37
Ci. freundii
Figure 3.3.1 - Dendrogram derived from profiles by the Random Amplification of Polymorphic
DNA (RAPD-PCR) generated with primers M13 and OPAA-10 of the 80 GNP strains isolated
from different raw milk samples. The profile grouping was done with the BioNumeric 5.0
software package, using the unweighted pair group method with arithmetic averages (UPGMA)
cluster analysis.
32
Results and discussion: Characterization of Gram-negative psychrotrophic bacteria
3.3.2 Phenotypic properties
According to their negative Gram reaction and the catalase and oxidase activities, the strains
were considered to be Pseudomonas and Enterobacteriaceae, and were specified by molecular
identification. Individual results of phenotypic characterization for Pseudomonas and
Enterobacteriaceae strains are listed in Tables 3.3.1 and 3.3.2, respectively. All the
Pseudomonas and Enterobacteriaceae isolates were catalase-oxidase positive and catalase
positive-oxidase negative, respectively. Enterobacteriaceae gave gas production, except for the
S. marcescens strains. Most psychrotrophic strains were actively motile after incubation, and
produced a different and distinctive pigmentation varying from cream/yellow/fluorescent
(pyoverdin pigment )/brown/blue (pyocyanin pigment) of Pseudomonas spp. to light pink/dark
red (prodigiosin pigment) of S. marcescens. P. fluorescens strains mostly produced
yellow/cream and blue pigments while P. aeruginosa strains showed yellow and brown/cream
pigmentation. The other psycrotrophic strains showed a gradation of colors from cream/yellow
to orange/brown.
Table 3.3.1 - Phenotypic properties of 63 Pseudomonas strains isolated from raw milk
Pseudomonas strains
PS34
PS20
PS86
PS65
PS26
PS27
PS38
PS44
PS32
PS42
PS53
PS87
PS29
PS67
PS59
PS16
PS19
PS23
PS24
PS14
PS64
PS60
P. aeruginosa
P. aeruginosa
P. aeruginosa
P. aeruginosa
P. aeruginosa
P. aeruginosa
P. aeruginosa
P. aeruginosa
P. aeruginosa
P. aeruginosa
P. aeruginosa
P. aeruginosa
P. aeruginosa
P. aeruginosa
P. aeruginosa
P. fluorescens
P. fluorescens
P. fluorescens
P. fluorescens
P. fluorescens
P. fluorescens
P. fluorescens
Growth in BHI
brotha
7 °C
+ (3)
+ (3)
+ (4)
+ (3)
+ (3)
+(23)
+ (3)
+ (1)
+ (2)
+ (6)
+ (8)
+ (3)
+(23)
+ (8)
+ (5)
+ (5)
+ (5)
+ (5)
+ (5)
+ (3)
+ (2)
+ (3)
22 °C
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
30 °C
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
33
Gas
production
Motility
Pigment
production
-
+
+
+
+
+
+
+
+
+
+
+
+
+
±0,5cm
+
+
+
±0,5cm
±0,5cm
Brown/cream
Yellow
Beige
Yellow/beige
Brown
Yellow/cream
Yellow
Brown/cream
Yellow
Brown/yellow
Yellow
Cream
Brown/yellow
Yellow
Brown/cream
Yellow
Yellow
Yellow/cream
Yellow/cream
Yellow
Yellow/cream
Yellow/cream
Results and discussion: Characterization of Gram-negative psychrotrophic bacteria
Pseudomonas strains
PS77
PS78
PS28
PS81
PS68
PS56
PS73
M3
M7
M37
M44
PR5
PS55
PS12
PS47
PS52
PS10
PS51
PS1
PS49
PS70
PS31
PS39
PS4
PS93
PS40
PS41
PS8
PS83
PS17
PS62
PS84
PS66
PS88
PS72
PS54
P. fluorescens
P. fluorescens
P. fluorescens
P. fluorescens
P. fluorescens
P. fluorescens
P. fluorescens
P. fluorescens
P. fluorescens
P. fluorescens
P. fluorescens
P. fluorescens
P. fragi
P. fragi
P. fragi
P. fulva
P. fulva
P. fulva
P. fulva
P. libanensis
P. mosselii
P. mosselii
P. mosselii
P. putida
P. putida
P. putida
P. putida
P. putida
P. putida
P. putida
P. rhodesiae
P. rhodesiae
Pseudomonas sp.
Pseudomonas sp.
Pseudomonas sp.
Pseudomonas sp.
Growth in BHI
brotha
7 °C
+ (5)
+ (6)
+ (3)
+ (5)
+ (9)
+ (6)
+ (2)
+ (2)
+ (5)
+ (3)
+ (1)
+ (2)
+ (3)
+ (4)
+ (5)
+ (5)
+(30)
+ (6)
+ (3)
+ (6)
+ (5)
+(23)
+ (1)
+(20)
+(12)
+ (6)
+ (1)
+(12)
+ (5)
+ (5)
+ (2)
+ (3)
+ (3)
+ (2)
+ (6)
+ (1)
22 °C
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (2)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
30 °C
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
+ (1)
34
Gas
production
Motility
Pigment
production
-
±0,5cm
±0,5cm
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
±0,5cm
+
+
+
+
Blue
Blue/brown
Cream/orange
Yellow/cream
Yellow/cream
Beige
Cream
Blue
Blue
Blue
Blue
Blue
Colorless
Cream
Yellow/cream
Cream
Cream
Yellow/cream
Dark yellow
Fluorescent
Yellow/cream
Brown/cream
Yellow/cream
Cream
Cream
Yellow
Dark yellow
Beige/cream
Yellow/cream
Yellow/cream
Pink/cream
Pink/cream
Cream
Yellow
Yellow/cream
Orange/yellow
Results and discussion: Characterization of Gram-negative psychrotrophic bacteria
Pseudomonas strains
Growth in BHI
brotha
7 °C
22 °C
Gas
production
Motility
Pigment
production
30 °C
PS61
PS13
PS75
PR3
PS11
Pseudomonas sp. + (1)
+ (1)
+ (1)
+
Fluorescent
Pseudomonas sp. + (3)
+ (1)
+ (1)
+
Yellow/brown
Pseudomonas sp. + (5)
+ (1)
+ (1)
±0,5cm
Cream
Pseudomonas sp. + (2)
+ (1)
+ (1)
Blue
P. teatrolens
+ (3)
+ (1)
+ (1)
Yellow
P. chlororaphis
PS6
subsp.
+ (5)
+ (1)
+ (1)
Orange
aurantiaca
a
Numbers in brackets ( ) indicate the days of incubation until growth of Pseudomonas strains at
different temperatures occurred
Table 3.3.2 - Phenotypic properties of 17 Enterobacteriaceae strains isolated from raw milk
Enterobacteriaceae
strains
Growth in BHI
brotha
7°C 22°C 30°C
Gas
production
Motility
Pigment
production
PS18 Cit. freundii
+ (4) + (1) + (1)
+
Brown/cream
PS37 Cit. freundii
+ (3) + (1) + (1)
+
+
Cream
PS25 Ent. cloacae
+ (5) + (1) + (1)
+
+
Colorless
PS57 H. alvei
+ (1) + (1) + (1)
+
+
Yellow/cream
PS85 H. alvei
+ (2) + (1) + (1)
+
+
Colorless
PS58 H. alvei
+ (1) + (1) + (1)
+
+
Yellow/cream
PS43 H. alvei
+ (1) + (1) + (1)
+
+
Brown/cream
PS46 H. alvei
+ (9) + (1) + (1)
+
+
Brown/cream
PS33 S. marcescens
+ (3) + (1) + (1)
+
Red/pink
PS69 S. marcescens
+ (5) + (1) + (1)
+
Red/pink
PS74 S. marcescens
+ (5) + (1) + (1)
+
Red/pink
PS90 S. marcescens
+ (5) + (1) + (1)
+
Red/pink
PS91 S. marcescens
+ (2) + (1) + (1)
+
Red/pink
PS92 S. marcescens
+ (3) + (1) + (1)
+
Red/pink
PS45 S. marcescens
+ (6) + (1) + (1)
+
Red/pink
PS36 S. marcescens
+ (2) + (1) + (1)
+
Red/pink
PS30 S. marcescens
+(18) + (1) + (1)
+
Red/pink
a
Number in brackets ( ) indicate the incubation time required until growth of
Enterobacteriaceae strains at different temperatures occurred
35
Results and discussion: Characterization of Gram-negative psychrotrophic bacteria
3.3.3 Growth and enzymatic activity at different temperatures
The growth ability of the 80 GNP strains was investigated at different temperatures (7, 22, 30
°C) over time to understand how storage conditions favoured or minimized their growth. For
both Pseudomonas and Enterobacteriaceae, growth was visible for all the strains at the tested
temperatures, as reported by other authors (Marchand et al., 2009), though growth rate differed
during incubation. With the exception of one strain, P. fulva grew faster at 30 °C than at 22 °C,
there were no detectable differences among the isolates in the growth test at 22 °C and 30 °C,
and growth occurred already after 24 hours of incubation (Tables 3.3.1 and 3.3.2). Most of the
strains were able to grow at a restricted temperature (7 °C) in the early days of incubation.
Regarding Pseudomonas spp., growth was generally visible from 24h (6 strains) to 6 days,
except for 10 strains showing lower growth rates for up to 30 days of incubation. Among the
Enterobacteriaceae, fast growth was observed in almost all the strains between 24h (3 strains of
H. alvei) and 6 days. Nine days were required to detect the growth of one of H. alvei, and one
strain of S. marcescens showed the lowest growth (18 days).
All psychrotrophic strains were also tested for the production of extracellular proteases, lipases
and lecithinases at 7, 22 and 30 °C up to 20 days. Individual results of enzymatic activities for
Pseudomonas and Enterobacteriaceae strains are listed in Tables 3.3.3 and 3.3.4, respectively.
Forty-one strains were found to be positive for all 3 enzymatic activities, and, P. fluorescens
stood out as the species with the most number of positive strains for all enzymatic activities at
the tested temperatures. Only a few data are available with regard to the influence of
temperature on psychrotroph enzymatic activity, and these are limited to protease.
The highest number of positive strains at all incubation temperatures was observed for lipolytic
activity (59/80), followed by proteolytic (31/80) and lechitinase activities (28/80). Enzymatic
activities varied among the Pseudomonas and Enterobacteriaceae strains and were markedly
influenced by incubation temperature, 30 °C being the optimal one.
With regard to protease production, P. fragi, P. putida, P. fulva and P. teatrolens species failed
to produce proteases at all incubation temperatures, as did 3 strains of P. aeruginosa and 1 of P.
fluorescens, P. mosselii and Pseudomonas sp.; among the Enterobacteriaceae, the production of
proteases was restricted to S. marcescens. The protease production of Pseudomonas strains was
studied by Marchand et al. (2009) who highlighted that P. fragi failed to produce proteases only
at 30 °C, and observed the lowest number of protease producing strains at 30 °C. Conversely, in
this study, the highest number of positive GNP for proteolytic activity was found at 30 and 22
°C 30 °C being the optimal temperature. Almost all the strains displayed marked proteolytic
activity within 2 days at 30 °C, and within 3 days at 22 °C. Twenty-seven out of 63 of the
Pseudomonas strains displayed protease production at 7 °C within at least 4 days, while four
out of the 17 Enterobacteriaceae strains were protease positive, starting from the sixth day at 7
°C.
Lipase production on tributyrin agar plates was faster at 30 °C, nevertheless 68 strains displayed
a noticeable lipolytic activity after incubation at 22 °C within 3 days; moreover 49 out of 63
Pseudomonas (77.77%) and 9 out of 17 Enterobacteriaceae (52.94%) strains showed lipase
production at 7 °C within 10 days of incubation. Three strains belonging to P. fluorescens and
one Pseudomonas sp. showed lipolytic activity after only 3 days at 7 °C, while it took at least 6
days for Enterobacteriaceae strains to produce lipolityc activity.
It was also observed that the number of Pseudomonas producing lecithinases increased with
incubation temperatures, from a 39.68% of positive strains at 7 °C to a 55.55% of lecithinaseproducing strains at 30 °C. On the other hand, almost half the Enterobacteriaceae (47%) were
able to degrade lecithin at 30 °C and 22 °C, whereas only 10% displayed lecithinase activity at
7 °C.
36
Results and discussion: Characterization of Gram-negative psychrotrophic bacteria
Our data only partially agree with Dogan and Boor (2003) who found no evidence of lipolysis
and lechitinase activity for P. putida strains, while the strains isolated in this study expressed
lipolytic activity.
3.3.4 Assessment of aprX gene in psychrotrophic strains
Proteolytic psychrotrophic bacteria are the main microorganisms responsible for the spoilage of
milk and milk products, due to their ability to produce thermostable proteases that hydrolyze
casein and decrease the yields and sensory qualities of dairy products (Sørhaug & Stepaniak,
1997). Pseudomonas are known for their production of metalloproteases in the cold chain of
raw milk (Craven & Macauley, 1992; Sorhaug & Stepaniak, 1997; Marchand et al., 2009). The
production of monomeric extracellular proteases of molecular weight varying between 23 kDa
and 56 kDa is a common feature of P. fluorescens. In this study the 80 GNP strains were
screened for the presence of aprX gene by aprX-PCR test, even though they did not exhibit
extracellular proteolytic activity when grown on skim milk agar. The aprX gene was detected in
19 out of 63 Pseudomonas strains, and was widespread among P. fluorescens (14/19), P.
rhodesiae (2/2), P. libanensis and P. chlororaphis subsp. aurantiaca (Table 3.3.3). Among the
Enterobacteriaceae, no amplification product was detected (Table 3.3.4), though all S.
marcescens strains were proteolytic.
Martins and others (2005) already illustrated the presence of the aprX gene in proteolytic
psychrotrophic bacteria isolated from raw milk. The apr gene, coding for heat-stable alkaline
metalloproteases with a pH optimum of 6.5–8.0 (Fairbairn & Law, 1986; Woods et al., 2001),
has been detected in proteolytic P. fluorescens, P. tolaasii, P. aeruginosa, Pseudomonas sp., S.
marcescens, and Flavobacterium-Cytophaga isolated from soil (Bach et al., 2001).
Table 3.3.3 - An overview of Pseudomonas strains: enzymatic characterization at 7, 22 and 30
°C and detection of aprX gene
Proteolysisa
Pseudomonas strains
b
7°C
c
22°C
d
b
30°C
Degradation of
lecithina
Lipolysisa
aprX
7°C
c
22°C
d
30°C
b
7°C
c
22°C
d
30°C
PS34
P. aeruginosa
-
-
-
-
+
(10)
+ (2)
+ (1)
-
-
-
PS20
P. aeruginosa
-
+(2)
+ (1)
-
+ (9)
+ (3)
+ (2)
+ (7)
+ (2)
+ (2)
PS86
P. aeruginosa
-
+ (2)
+ (1)
-
-
+ (3)
+ (1)
-
-
-
PS65
P. aeruginosa
+ (5)
+ (3)
+ (1)
-
+ (7)
+ (3)
+ (3)
+ (6)
-
-
PS26
P. aeruginosa
-
-
-
-
-
-
-
-
+ (3)
+ (2)
PS27
P. aeruginosa
-
+ (1)
+ (1)
-
+ (6)
+ (1)
+ (1)
-
+ (2)
+ (1)
PS38
P. aeruginosa
-
+ (1)
+ (1)
-
+ (9)
+ (2)
+ (2)
-
+ (2)
+ (1)
PS44
P. aeruginosa
-
-
-
-
-
+ (6)
+ (3)
-
-
-
PS32
P. aeruginosa
-
+ (2)
+ (1)
-
+ (9)
+ (2)
+ (1)
-
+ (2)
+ (1)
PS42
P. aeruginosa
-
+ (2)
+ (1)
-
-
+ (2)
+ (1)
-
-
-
PS53
P. aeruginosa
-
+ (1)
+ (1)
-
+ (7)
+ (1)
+ (1)
-
+ (2)
+ (1)
PS87
P. aeruginosa
-
+ (2)
+ (1)
+
-
+ (4)
+ (1)
-
-
-
PS29
P. aeruginosa
-
+ (2)
+ (1)
-
+ (9)
+ (3)
+ (3)
-
-
-
PS67
P. aeruginosa
-
+ (2)
+ (1)
-
+ (8)
+ (2)
+ (1)
-
+ (2)
+ (2)
37
Results and discussion: Characterization of Gram-negative psychrotrophic bacteria
Proteolysisa
Pseudomonas strains
b
7°C
c
22°C
d
b
30°C
Degradation of
lecithina
Lipolysisa
aprX
7°C
c
22°C
d
30°C
b
7°C
c
22°C
d
30°C
PS59
P. aeruginosa
-
-
-
-
-
+ (4)
+ (2)
-
+ (3)
+ (2)
PS16
P. fluorescens
+ (7)
+ (2)
+ (1)
+
+ (7)
+ (2)
+ (2)
+ (6)
+ (3)
+ (1)
PS19
P. fluorescens
+ (6)
+ (3)
+ (1)
+
+ (6)
+ (1)
+ (1)
+(11)
+ (5)
+ (3)
PS23
P. fluorescens
-
+ (2)
+ (1)
+
+ (9)
+ (2)
+ (2)
+ (6)
+ (6)
+ (2)
PS24
P. fluorescens
+ (5)
+ (6)
+ (2)
+
+ (7)
+ (1)
+ (1)
+ (6)
+ (2)
+ (2)
PS14
P. fluorescens
+ (5)
+ (3)
+ (2)
+
+ (6)
+ (1)
+ (1)
+ (7)
+ (6)
+ (2)
PS64
P. fluorescens
+ (7)
+ (2)
+ (1)
+
+ (5)
+ (3)
+ (3)
+ (6)
+ (2)
+ (1)
PS60
P. fluorescens
+ (5)
+ (2)
+ (2)
-
+ (6)
+ (2)
+ (2)
+(10)
+ (2)
+ (2)
PS77
P. fluorescens
+(10)
+ (5)
+ (3)
-
+ (6)
+ (2)
+ (3)
+ (7)
+ (5)
+ (5)
PS78
P. fluorescens
+(10)
+ (3)
+ (2)
+
+ (6)
+ (2)
+ (3)
+(11)
+ (5)
+ (3)
PS28
P. fluorescens
+(10)
+ (3)
+ (2)
+
+ (7)
+ (2)
+ (1)
-
-
-
PS81
P. fluorescens
+(10)
+ (2)
+ (2)
+
+ (4)
+ (2)
+ (1)
-
-
-
PS68
P. fluorescens
-
-
-
-
+ (6)
+ (2)
+ (1)
-
-
-
PS56
P. fluorescens
+ (7)
+ (3)
+ (2)
-
+ (7)
+ (2)
+ (3)
+ (7)
+ (2)
+ (2)
PS73
P. fluorescens
+ (7)
+ (3)
+ (2)
+
+ (8)
+ (2)
+ (2)
-
-
-
M3
P. fluorescens
+ (8)
+ (2)
+ (2)
+
+ (3)
+ (1)
+ (2)
+(12)
+ (4)
+ (1)
M7
P. fluorescens
+ (8)
+ (2)
+ (1)
+
+ (3)
+ (1)
+ (1)
+ (8)
+ (4)
+ (2)
M37
P. fluorescens
+ (5)
+ (2)
+ (1)
+
+ (6)
+ (1)
+ (1)
+ (7)
+ (3)
+ (2)
M44
P.fluorescens
+ (5)
+ (2)
+ (2)
+
+ (3)
+ (1)
+ (1)
+ (7)
+ (3)
+ (1)
PR5
P. fluorescens
+ (6)
+ (2)
+ (1)
-
+ (6)
+ (2)
+ (3)
+(10)
+ (4)
+ (2)
PS55
P. fragi
-
-
-
-
+ (9)
+ (2)
+ (2)
-
+ (4)
+ (2)
PS12
P. fragi
-
-
-
-
+ (6)
+ (2)
+ (2)
-
-
-
PS47
P. fragi
-
-
-
-
+ (6)
+ (2)
+ (1)
-
-
-
PS52
P. fulva
-
-
-
-
+(10)
+ (3)
+ (3)
-
-
-
PS10
P. fulva
-
-
-
-
+(10)
+ (3)
+ (2)
-
-
-
PS51
P. fulva
-
-
-
-
+ (9)
+ (6)
+ (3)
-
-
-
PS1
P. fulva
PS49
P. libanensis
PS70
P. mosselii
-
PS31
P. mosselii
+(10)
PS39
P. mosselii
+(10)
PS4
P. putida
PS93
P. putida
PS40
PS41
PS8
-
-
-
-
+(10)
+ (6)
+ (3)
-
-
-
+ (7)
+ (2)
+ (1)
+
+ (6)
+ (2)
+ (3)
+ (8)
+ (3)
+ (2)
-
-
-
-
+ (1)
+ (1)
+(10)
+ (1)
+ (1)
+ (2)
+ (1)
-
+ (9)
+ (1)
+ (1)
-
+ (2)
+ (1)
+ (1)
+ (1)
-
-
+ (1)
+ (1)
+(13)
+ (1)
+ (1)
-
-
-
-
-
+ (3)
+ (1)
-
-
-
-
-
-
-
-
+ (5)
+ (3)
-
+ (3)
+ (1)
P. putida
-
-
-
-
+(10)
+ (2)
+ (3)
-
-
-
P. putida
-
-
-
-
+ (8)
+ (2)
+ (1)
-
-
-
P. putida
-
-
-
-
-
+ (3)
+ (1)
-
-
-
38
Results and discussion: Characterization of Gram-negative psychrotrophic bacteria
Proteolysisa
Pseudomonas strains
b
7°C
-
c
22°C
PS83
P. putida
-
PS17
P. putida
-
-
PS62
P. rhodesiae
+ (5)
+ (2)
PS84
P. rhodesiae
+ (5)
+ (2)
PS66
Pseudomonas sp.
-
+ (2)
PS88
Pseudomonas sp.
-
+ (2)
PS72
Pseudomonas sp.
-
PS54
Pseudomonas sp.
-
PS61
Pseudomonas sp.
-
PS13
Pseudomonas sp.
+ (4)
PS75
Pseudomonas sp.
PR3
Pseudomonas sp.
d
b
30°C
-
Degradation of
lecithina
Lipolysisa
aprX
7°C
c
22°C
d
30°C
b
7°C
-
c
22°C
30°C
-
+ (8)
+ (3)
+ (3)
-
-
+ (7)
+ (3)
+ (1)
-
-
-
+ (1)
+
+ (9)
+ (2)
+ (3)
+ (7)
+ (2)
+ (2)
+ (1)
+
+ (7)
+ (3)
+ (1)
+ (7)
+ (5)
+ (2)
+ (1)
-
-
+ (3)
+ (2)
-
-
-
+ (1)
-
+(10)
+ (4)
+ (1)
-
-
-
-
-
-
-
+ (2)
+ (1)
-
-
-
+ (2)
+ (2)
-
+ (8)
+ (2)
+ (2)
+(10)
+ (2)
+ (2)
+ (5)
+ (2)
-
+ (8)
+ (5)
+ (2)
-
+ (5)
+ (3)
+ (1)
+ (1)
-
+ (6)
+ (2)
+ (1)
+(10)
+ (1)
+ (1)
+ (7)
+ (2)
+ (1)
-
+ (8)
+ (2)
+ (2)
-
-
-
+ (5)
+ (2)
+ (4)
-
+ (3)
+ (2)
+ (3)
-
-
-
-
-
+ (2)
+ (2)
PS11
P. teatrolens
+ (6)
+ (2)
P. chlororaphis
PS6
+ (5)
+ (2)
+ (1)
+
+ (5)
+ (2)
+ (1)
+ (7)
subsp. aurantiaca
a Numbers in brackets ( ) indicate the incubation time required to visualize enzymatic activities
b
Proteolysis and lipolysis were visualized up to 10 days and degradation of lecithin up to 20 days
c
Proteolysis , lipolysis and degradation of lecithin were visualized up to 7 days
d
Proteolysis and lipolysis were visualized up to 3 days and degradation of lecithin up to 5 days
39
-
d
-
Results and discussion: Characterization of Gram-negative psychrotrophic bacteria
Table 3.3.4- An overview of Enterobacteriaceae strains: enzymatic characterization at 7, 22 and
30 °C and detection of aprX gene
Enterobacteriaceae
strains
Proteolysisa
b
7°C
c
22°C
Lipolysisa
aprX
d
b
30°C
7°C
c
22°C
Degradation of lecithina
d
30°C
b
7°C
c
22°C
d
30°C
PS18
Cit. freundii
+ (4)
+ (1)
+ (1)
+
-
-
-
-
-
-
PS37
Cit. freundii
+ (3)
+ (1)
+ (1)
+
-
+ (3)
+ (1)
-
-
-
PS25
Ent .cloacae
+ (5)
+ (1)
+ (1)
+
-
-
-
-
-
-
PS57
H. alvei
+ (1)
+ (1)
+ (1)
+
+ (6)
+ (2)
+ (3)
-
-
-
PS85
H. alvei
+ (2)
+ (1)
+ (1)
+
+ (7)
+ (3)
+ (1)
-
-
-
PS58
H. alvei
+ (1)
+ (1)
+ (1)
+
+ (6)
+ (1)
+ (2)
-
-
-
PS43
H. alvei
+ (1)
+ (1)
+ (1)
+
-
-
-
-
-
-
PS46
H. alvei
+ (9)
+ (1)
+ (1)
+
+ (6)
+ (2)
+ (3)
-
-
-
PS33
S. marcescens
+ (3)
+ (1)
+ (1)
-
+ (9)
+ (1)
+ (2)
-
+ (1)
+ (1)
PS69
S. marcescens
+ (5)
+ (1)
+ (1)
-
-
+ (2)
+ (2)
+(12)
+ (2)
+ (2)
PS74
S. marcescens
+ (5)
+ (1)
+ (1)
-
+(10)
+ (2)
+ (3)
+(12)
+ (1)
+ (1)
PS90
S. marcescens
+ (5)
+ (1)
+ (1)
-
-
+ (2)
+ (2)
+(10)
+ (1)
+ (1)
PS91
S. marcescens
+ (2)
+ (1)
+ (1)
-
-
+ (3)
+ (1)
-
+ (3)
+ (2)
PS92
S. marcescens
+ (3)
+ (1)
+ (1)
-
+ (6)
+ (3)
+ (1)
+ (7)
+ (5)
+ (2)
PS45
S. marcescens
+ (6)
+ (1)
+ (1)
-
+(10)
+ (2)
+ (2)
-
+ (1)
+ (1)
PS36
S. marcescens
+ (2)
+ (1)
+ (1)
-
-
+ (3)
+ (2)
-
+ (4)
+ (2)
-
-
PS30 S. marcescens
+(18)
+ (1)
+ (1)
+ (8)
+ (2)
+ (3)
a Numbers in brackets ( ) indicate the incubation time required to visualize enzymatic activities
b
Proteolysis and lipolysis were visualized up to 10 days and degradation of lecithin up to 20 days
c
Proteolysis , lipolysis and degradation of lecithin were visualized up to 7 days
d
Proteolysis and lipolysis were visualized up to 3 days and degradation of lecithin up to 5 days
40
Results and discussion: Characterization of Gram-negative psychrotrophic bacteria
3.4 Conclusions
Psychrotrophic bacteria play a major role in the spoilage of cold milk and dairy products, and
their proteolytic and lipolytic enzymes constitute one of the main limiting factors for
maintaining the technological and sensory quality of milk. The presence of GNP bacteria in raw
milk, bacteria harbouring spoilage features, and the predominance of Pseudomonas spp. and
Enterobacteriaceae have been evidenced in Lombardy raw milk, where high biodiversity is
highlighted. This study provides a better understanding of the outgrowth of GNP bacteria in
milk, and the different enzymatic traits that are demonstrated to be more influenced by
temperature conditions than growth rate. A strict control of storage temperature, along with the
reduction of GNP contamination, can constitute an effective control measure against spoilage
events. Indeed, the prevention of deterioration in processed milk and dairy products calls for a
rapid detection of enzymes exhibiting high protein and lipid degrading effects in raw milk.
Therefore more research is needed towards the early detection of spoilage phenomena.
3.5 References
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Results and discussion: Characterization of Gram-negative psychrotrophic bacteria
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Results and discussion: Characterization of Gram-negative psychrotrophic bacteria
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production of pasteurised milk, evaluated by randomly amplified polymorphic DNA (RAPD)
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raw cow’s milk. Food Microbiol 26:228-231.
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PF, eds., Academic Press, London, 2002, pp. 1564–1568.
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Rosenberg E, Schleifer KH, Stackebrandt E, eds., Springer Verlag, Berlin, 2006, pp. 219–244.
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Spencer de ALR, eds., Humana Press Inc, Totowa, New Jersey, NJ, 2001, pp. 3–11.
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probe from bacteriophage M13. J Bacteriol 171:2528–2532.
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43
Results and discussion: Characterization of Gram-negative psychrotrophic bacteria
Ma Y et al., 2000, Effects of somatic cell count on quality and shelf-life of pasteurized fluid
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products. A review of basic information and practical aspects. Kieler Milchwirtchaftliche
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isolated from refrigerated raw milk. Int J Food Microbiol 102:203– 211.
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bacteria isolated from refrigerated raw milk. Int J Food Microbiol 111:144-148.
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farmhouse goats’ milk cheeses from Sierra de Aracena. Food Microbiol 26:294-304.
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Pseudomonas and Bacillus isolates. J. Mol. Catal., B Enzym. 40:127–131.
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of species of the genus Pseudomonas (sensu stricto) and estimation of the natural intrageneric
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Nam HM et al., 2010, In vitro activities of antimicrobials against six important species of gramnegative bacteria isolated from raw milk samples in Korea. Foodborne Pathog Dis 7:221-224.
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refrigerated raw milk. Int J Food Microbiol 63:41-46.
44
Results and discussion: Characterization of Gram-negative psychrotrophic bacteria
Raats D et al., 2011, Molecular analysis of bacterial communities in raw cow milk and the
impact of refrigeration on its structure and dynamics. Food Microbiol 28:465–471.
Rasolofo EA et al., 2010, Molecular analysis of bacterial population structure and dynamics
during cold storage of untreated and treated milk. Int J Food Microbiol 138:108–118.
Ray B. 2004, Fundamental Food Microbiology. 3rd edn. Boca Raton, Florida: CRC Press.
Sajben E et al., 2011, Characterization of pseudomonads isolated from decaying sporocarps of
oyster mushroom. Microbiol Res 166:255–267.
Shah NP. 1994, Psychrotrophs in milk: a review. Milchwissenschaft, 49:432–437.
Sørhaug T, Stepaniak L, 1997, Psychrotrophs and their enzymes in milk and dairy products:
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Suarez B, Ferreiros CM, 1991, Psychotrophic flora of raw milk: resistance to several common
disinfectants. J Dairy Res 58:127-136.
Tornadijo ME et al., 1993, Study of Enterobacteriaceae throughout the manufacturing and
ripening of hard goats’cheese. J Appl Bacteriol 75:240-246.
Tornadijo ME et al., 2001, Study of Enterobacteriaceae during the manufacture and ripening of
San Simón cheese. Food Microbiol 18:499–509.
Woods RG et al., 2001, The aprX–lipA operon of Pseudomonas fluorescens B52: a molecular
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45
Results and discussion: Characterization of VOCs in milk contaminated with psychrotrophs
3.6 Characterization of volatile compounds in milk contaminated with psychrotrophic
bacteria by SPME gas chromatography-mass spectrometry
Psychrotrophic Gram-negative bacteria, mostly Pseudomonas spp. and Enterobacteriaceae, play
a leading role in spoilage of milk and dairy products (Walker, 1988; Sorhaug & Stepaniak,
1997; Morales et al., 2003; Chaves-Lopez et al., 2006). The main source of psychrotrophic
bacteria that become predominant during the refrigerated storage of raw milk, is inadequately
sanitized milking equipment (Cousin, 1982) and common sources of post-pasteurization
contamination by psychrotrophs are improperly cleaned pasteurizers and filling machines
(Gruetzmacher & Bradley, 1999). In milk and dairy products spoilage strongly depends on the
production of heat-resistant extracellular enzymes by psychrotrophic bacteria. Mainly proteases
and lipases are capable of releasing undesired metabolic compounds arising from nutrient
degradation at low temperatures and responsible for off-flavours in milk. Proteases that degrade
casein, are associated with textural changes (structural defects) in milk such as gelation and
increased viscosity and with unclean and bitter flavours in cheese and other dairy products. On
the other hand, rancid, soapy and fruity aromas (flavour defects) are caused by lipases due to
the fat breakdown in cream, butter, cheese and UHT products. Literature references have
reported VOCs profile of raw (Bassette et al., 1966; Vazquez-Landaverde et al., 2005;
Vazquez-Landaverde et al., 2006; Hettinga et al., 2008) pasteurized and sterilized (Contarini et
al., 1997; Valero et al., 1999; Valero et al., 2001; Contarini & Povolo, 2002) milk samples. Offflavour development at a given storage time, usually at the end of shelf-life of the product,
(Urbach & Milne, 1987; Jenq et al., 1988; Cormier et al., 1991; Leong et al., 1992; VallejoCordoba & Nakai, 1994; Marsili, 1999b; Contarini & Povolo, 2002) and VOCs changes
affected by packaging material (Karatapanis et al., 2006) in milk have also been studied. Recent
studies reported the production of volatile compounds associated with bacteria isolated from
different foodstuffs, such as beef (Ercolini et al., 2009) and shrimps (Jaffrès et al., 2011) and
seven different species of Pseudomonas of dairy origin were screened for their VOC production
in cheese (Morales et al., 2005) but very limited information exists in the literature on changes
in flavour compounds of milk spoiled with different psychrotrophic bacteria. In addition,
studies on the correlation between spoilage-related molecules release and the development of
specific microbial species in milk are still not available. Pseudomonas is the psychrotrophic
genus of greatest concern with respect to the spoilage of milk and dairy products (Ternström et
al., 1993; Ralyea et al., 1998). Enterobacteriaceae have also been frequently encountered in
milk and during cheese production (Sorhaug & Stepaniak, 1997; Morales et al., 2003; Mounier
et al., 2005; Chaves-Lopez et al., 2006), and they are considered as a negative flora (i.e. marker
for hygiene, cheese texture defects, blowing and off-flavours). The purpose of the second study
of this PhD thesis was to investigate the spoilage potential of lipo-proteolytic psychrotrophic
bacteria, previously isolated from raw milk (Decimo et al., 2014), assessing their volatile
fraction using solid-phase microextraction (SPME) and gas chromatography coupled to mass
spectrometry (GC-MS). A further aim of this work was to identify molecular markers for the
early detection of milk spoilage.
3.7 Material and methods
3.7.1 Bacterial strains and milk contamination
The strains tested in this study had previously been isolated from raw milk samples, identified
and characterized for their enzymatic activity at different temperatures (Decimo et al., 2014).
Since the spoilage potential of bacteria is mainly related to their lipo-proteolytic activity, five
46
Results and discussion: Characterization of VOCs in milk contaminated with psychrotrophs
psychrotrophic strains, showing enzymatic activities both at 7 and 22°C, except for P. mosselii
PS39 that was negative for lipase production at 7°C, were selected for the milk contamination
and VOC monitoring experiments as follows.
Aliquots of 200 mL of whole UHT milk were spiked with 106 CFU mL -1 of an overnight BHI
cultures of each of the following psychrotrophic strains incubated at 30 °C: Pseudomonas
fluorescens PS14, Ps. fragi PS55, Ps. mosselii PS39, Ps. rhodesiae PS62 and Serratia
marcescens S92. This inoculum level was in chosen since bacteria need to reach a similar
population before significant proteo-lipolytic activity occurs (Cogan, 1980; Outtara et al.,
2004). The control sample of UHT milk was incubated along with the samples contaminated
with psychrotrophic bacteria at 10°C for 5, 10, 20 days and at 5°C for 20 days.
3.7.2 VOC determination by SPME/GC-MS
Volatile compounds in UHT milk were analysed using a gas chromatography device, model GC
6850, Agilent (Agilent Technologies, Barcelona, Spain) coupled with a mass spectrometer 5975
C VL (Agilent) series GC/MSD with the Triple-Axis Detector (TAD) after solid-phase
microextraction.
Briefly, spoiled UHT milk samples (7 ml) were heated in 15 ml vials sealed with polypropylene
screw-on caps and PTFE/silicone septa (Supelco Bellefonte, PA, USA) for 15 min at 45°C to
equilibrate the system.Volatiles were extracted from the headspace of the vial for 30 min with a
75 µm carboxen/polydimethylsiloxane (CAR/PDMS) SPME fiber (Supelco). Peak separation
was carried out on a 60 m length × 0.25 mm internal diameter × 1 µm film thickness column
(Quadrex 007-5MS-60-1.0, DB5, Supelco). Volatile compounds were analyzed following the
gas chromatographic conditions described by Contarini and Povolo (2002), modifying MS
settings and oven temperature. Briefly thermal desorption of volatile compounds was carried
out by keeping the SPME fiber in the split/splitless injector at 270°C for 3 min in splitless
mode; oven temperature was held at 40°C for 8 min, programmed to 150°C at a rate of
4°C/min, and held at 150°C for 10 min. Helium was used as a carrier gas at a flow rate of 1.0
ml/min. MS temperature adopted were as follows: interface, 270°C; source, 230°C; quadrupole,
150°C; acquisition was performed in electron impact (EI) mode (70 eV) and mass range used
was m/z 33-300.
Peak identification of compounds was conducted by comparing their retention times and the
collected ion (mass) spectra to the reference spectral library, NIST MS search 2.0 library
(NIST; Perkin–Elmer; Waltham, MA). An 85-90% level of certainty in the spectral match
criteria was used as a cut-off for spectral identification. The limit of detection (LOD) of a
compound was considered to be the minimum concentration at which it could be identified by
its mass spectrum, retention time and with a relative abundance of at least 3 times the signal-tonoise ratio. The peak areas of volatile compounds were taken to be their relative abundances
and were estimated as mean value of duplicate analysis for each sample.
3.7.3 Sensory evaluation
Descriptive aroma analysis was performed on milk samples stored at 10°C after 10 day of
incubation. Descriptive sensory test was performed to differentiate between the aroma of milk
spoiled by the 5 different psychrotrophic strains. Samples was evaluated by a panel of 10 tasters
(7 females and 3 males) selected from the members of the Department of Nutrition, Food
Science and Technology of the Complutense University of Madrid. The panellists were
previously trained in the sensory assessment of dairy products. During the training, reference
models (rotten egg, cut grass, fruity, sweaty, burnt milk or pudding, sulphur, metallic, baby
47
Results and discussion: Characterization of VOCs in milk contaminated with psychrotrophs
vomit, rancid, pungent, alcohol or acetic acid, unpleasant buttery, cooked) were prepared in
order to familiarise the testers with the expected flavours resulting from psychrotrophic
contamination of milk. Before sensory analysis, the samples were kept to temperate for 15 min
at room temperature. For evaluation, 100 ml of milk were placed into 250-ml Pirex sniff-top
bottles wrapped in aluminum foil. The bottles were wrapped in aluminium foil to prevent
panelists from visually assessing the samples. Panellists were also asked to give information
about the rate of coagulation of milk after sniffing samples.
3.8 Results and discussion
3.8.1 VOC determination of spoiled UHT milk samples during storage
Qualitative data were obtained by calculating the area of peak relative to the quantifier ion of
the mass spectrum for each of the VOC. If the relative abundance of VOCs ranged from 7 to 8
log peak Area, the corresponding compounds were considered highly produced (++), on the
other hand if the peak measurements resulted between 6,5-5 log Area, these compounds showed
a medium production (+) while if the VOC area values were less than 4 log Area, the
corresponding compounds were flagged as detected in trace amount (t)
In Figure 3.8.1 is shown an example of an SPME-GC/MS metabolite profile obtained for the
UHT milk spiked with P. fluorescens PS14 at 10°C after 10 days of storage.
10
600000
600000
550000
500000
500000
Abundance
450000
400000
400000
350000
1
17
300000
300000
250000
200000
200000
11
8
9
6
150000
2
14
5
100000
16
100000
50000
3 4
10.00
10
12
7
15.00
15
20.00
20
25.00
25
13
15
30.00
30
18
19
20
35.00
35
21
40.00
40
22
45
Time (min)
Figure 3.8.1 SPME-GC/MS chromatogram of UHT milk contaminated with P. fluorescens
PS14. Peaks: (1) acetone; (2) dimethylsulphide; (3) hexane; (4) 2,3 butanedione; (5) 2butanone; (6) 2-pentanone; (7) pentanal; (8) mercaptoacetone, (9) 3-methylbutanol;(10)
disulphide dimethyl; (11) butanoic acid, (12) hexanal; (13) dimethyl sulfoxide; (14) 2heptanone; (15) heptanal; (16) dimethyl sulfone; (17) hexanoic acid; (18) 5-hepten-2one-6methyl; (19) benzaldehyde; (20) octanal;(21) 2-nonanone; (22) octanoic acid.
48
Results and discussion: Characterization of VOCs in milk contaminated with psychrotrophs
Fiftysix VOCs belonging to 7 chemical groups (aldehydes, ketones, fatty acids, esters, alcohols,
sulphur compounds and hydrocarbons) were identified.
VOCs detected in control UHT milk before incubation (Table 3.8.1, T0) were acetone, butan-2one, pentan-2-one, pentanal, 2,3,3-trimethyl pentane, methyldisulfanylmethane, hexanal,
heptan-2-one, methylsulfonylmethane, benzaldehyde. Methyl ketones with 3, 4, 5 and 7 carbon
atoms, resulted the main group of volatile compounds as was reported for UHT milk at day 0 of
storage by Valero et al. (2001) and for volatile fraction of direct UHT milk by Contarini and
Povolo (2002). Whereas acetone and butan-2-one are supposed to be derived from bovine
metabolism (Bassette et al., 1966; Gordon & Morgan, 1972; Urbacch & Milne, 1987), pentan-2one and heptan-2-one are in part thermally-induced products, arising from β-ketoacid
decarboxylation (Calvo & de la Hoz, 1992); they can also formed by β-oxidation of fatty acids,
followed by decarboxylation (Grosch, 1982). As reported by Contarini and Povolo (2002)
methyl ketones seemed to have the highest correlation to the severity of heat treatment on milk,
being heptan-2-one a suitable marker for heat processing. Regarding aldehydes, the presence of
pentanal and hexanal could be due to light exposure or Cu oxidation (Cadwallader & Howard,
1998; Marsili & Miller, 1998) or to oxidation of unsatured fatty acids whereas benzaldehyde
could be generated from phenylalanine (Nursten, 1997). Differently from our data, Valero et al.
(2001) found that the main aldehydes in UHT milk were acetaldehyde, butanal, hexanal, 2methyl butanal, 3-methyl butanal and benzaldehyde. Methyldisulfanylmethane and
methylsulfonylmethane probably derived from methionine (Dumont & Adda, 1979) and have a
strong contribution (along with methyl ketones) to the aroma of UHT milk (Badings, 1984) and
have also been related to the intensity of thermal treatment (Bosset et al., 1994). VazquezLandaverde et al. (2006) reported that whole UHT milk contained substantially high
concentrations of hydrogen sulfide, methanethiol, carbon disulfide, dimethyl trisulfide, and
dimethyl sulfoxide being methanethiol, dimethyl trisulfide and dimethyl sulfide the most
important contributors to the sulfurous note in milk. The 2,3,3-trimethyl pentane hydrocarbon
was identified in this study in the headspace of control UHT milk sample whereas 2,2,4trimethyl pentane was found in whole pasteurized milk packaged in different material during
storage time by Karatapanis et al. (2006).
As shown in Table 3.8.1, a general trend to VOCs production during the storage time at 10°C
was observed in control UHT milk sample. The formation of carbon disulphide, heptanal, 6methylhept-5-en-2-one was highlighted after 5 days of incubation while hexane (trace amount),
3-methylbutan-1-ol (t), pentan-1-ol (t), 2,2-dimethyl heptane, nonan-2-one, octanal (t) were
formed after 10 days and methylsulfanylmethane, butane-2,3-dione were found only after 20
days. Whereas some compounds such as alcohols, aldehydes, sulphur compounds, and
hydrocarbons detected at 10°C, was not noticed in same samples stored at 5°C after 20 days.
49
Results and discussion: Characterization of VOCs in milk contaminated with psychrotrophs
Table 3.8.1 VOCs detected in UHT milk at T0 and during storage time (days) at 10°C and 5°C
Storage time (d) at 10°C
5
10
20
Storage time (d) at 5°C
20
VOCs
T0
Alcohols
3-Methylbutan-1-ol
Pentan-1-ol
nd
nd
nd
nd
t
t
t
+
nd
nd
Aldehydes
Pentanal
Hexanal
Heptanal
Octanal
Benzaldehyde
+
+
nd
nd
+
+
+
+
nd
+
+
+
+
nd
+
+
+
+
b
t
+
+
+
nd
t
+
+
nd
+
+
+
nd
nd
+
nd
+
+
+
+
nd
+
nd
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
nd
nd
+
+
nd
+
+
+
nd
+
+
+
+
+
+
+
nd
nd
+
+
+
+
+
t
+
+
nd
+
nd
Ketones
Acetone
Butane-2,3-dione
Butan-2-one
Pentan-2-one
Heptan-2-one
6-Methylhept-5-en-2-one
Nonan-2-one
Sulphur compounds
Methylsulfanylmethane
Carbon-disulphide
Methyldisulfanylmethane
Methylsulfonylmethane
a
Hydrocarbons
Hexane
nd
nd
2,3,3-Trimethylpentane
+
+
2,2-Dimethyl heptane
nd
nd
a
nd, not detected
b
t, molecole occuring in trace amount
The results of the VOC analysis of the milk samples spoiled with five different psychrotrophic
bacteria during the storage time at 10°C and 5°C are reported in Tables 3.8.2 and 3.8.3.
Qualitative data indicated that the VOCs patterns of spoiled milk samples were partially
identical and considerable differences were found concerning the evolution of VOCs production
during the storage time.
Seven ketones (acetone, butan-2-one, pentan-2-one, heptan-2-one, 6-methylhept-5-en-2-one,
nonan-2-one, butane-2,3-dione), 4 fatty acids (acetic, butanoic, hexanoic, octanoic acids), 4
sulphur
compounds
(methylsulfanylmethane,
methyldisulfanylmethane,
methylsulfonylmethane, methylsulfinylmethane), 4 aldehydes (exanal, heptanal, octanal,
benzaldehyde) and 2 alcohols (2-methylpropan-1-ol, 3-methylbutan-1-ol) were detected in all
50
Results and discussion: Characterization of VOCs in milk contaminated with psychrotrophs
spoiled milk samples at both incubation temperatures (Tables 3.8.2 and 3.8.3), whereas others,
distinctive of specific strains, were produced only at 10 °C.
With regard to VOC production during storage at 10 °C the progressive formation of new
volatile compounds, mostly sulphur compounds, fatty acids, esters and alcohols was evident
from 5 to 20 days. Among the predominant alcohols found in spoiled milk, 2-methylpropan-1ol was produced by P. fragi, P. rhodesiae and S. marcescens at all sampling times and it was
detectable in milk spiked with P. mosselii after 10 days of storage whereas it was always absent
in the headspace of UHT control sample and of milk contaminated with P. fluorescens. 3methylbutan-1-ol, occurred in trace amount in UHT control samples from the 10th day of
storage, was produced in milk samples contaminated with P. fragi, P. mosselii, P. rhodesiae
and S. marcescens already after 5 days, while the branched-chain alcohol was present only in
minimal traces after 5 days of incubation in milk spoiled with P. fluorescens PS14 and
increased during the storage time, being appreciable after 10 days. Morales et al. (2005)
reported that 3-methylbutan-1-ol was mainly produced by P. libanensis and P. rhodesiae in
laboratory-scale cheeses and ethanol, 3-methylbutan-1-ol and 2-methylpropan-1-ol were the
predominant alcohols in cheeses inoculated with Enterobacteriaceae (Morales et al., 2004).
Regarding aldehydes production, no significant differences were recorded between UHT
control and milk samples contaminated with psychrotrophic strains. Octanal, only found in
minimal traces at the end of storage of UHT control milk, showed a small peak in all
chromatograms of the spoiled milk. Valero et al. (2001) reported the production of octanal not
in whole but only in skimmed UHT milk in low concentrations (starting) from 45 days of
storage at 25°C as a result of lipid oxidation. Concerning ketones, butane-2,3-dione (diacetyl)
production was recorded in all spoiled milk samples at different storage times. P. rhodesiae, S.
marcescens and P. mosselii were the strongest producing-species since the 5th day of
incubation; diacetyl was detected in the headspaces of milk contaminated with P. fluorescens
and P. fragi after 10 and 20 days of storage at 10 °C, respectively. Strangely, butan-2-one was
found in minimal traces in milk spoiled with S. marcescens after 5 and 10 days of storage,
despite its important presence in UHT control milk. The ketone butan-2-one could be derived
from butane-2,3-dione, which is produced by fermentation of lactose and metabolism of citrate.
The reduced amounts of butan-2-one by Serratia marcescens could be attributed to the
variations of the enzyme activity in metabolic pathway. Nonan-2-one was almost absent only in
UHT control sample and in milk spoiled with P. fragi and P. mosselii after 5 days of storage.
Among sulphur compounds methylsulfanylmethane, showing a low detection level in UHT
milk control, was the major sulphur compound found in the headspace of all spoiled milk
samples at all incubation time. In particular it occurred in trace amount in P. fluorescens spoiled
milk after 5 days of storage while it was generated in appreciable amount in all other
contaminated samples during incubation time. P. rhodesiae and S. marcescens showed
formation of the methylsulfinylmethane already after 5 days of storage while no production of
this sulphur compound was found in unspoiled UHT milk during the whole storage time as well
as in milk spoiled with P. fluorescens and P. fragi till 5 and 10 days of storage, respectively. No
fatty acids were found in the headspaces of UHT control sample during storage time and in milk
spoiled with P. fragi after 5 and 10 of storage at 10 °C. P. fluorescens released butanoic and
hexanoic acids at all storage times while acetic and octanoic acids were detected after 20 and 5
days of storage, respectively. P. mosselii produced butanoic acid already after 5 day of storage,
hexanoic and octanoic acids after 20 days and acetic acid was identified with a small peak only
at the end of storage. P. rhodesiae and S marcescens seemed to be the strongest acid-producing
species, showing an early appearance of all four fatty acids during storage.
51
Results and discussion: Characterization of VOCs in milk contaminated with psychrotrophs
Qualitative data indicated that each contamination resulted in complex and specific VOC
profiles of the milk samples during the storage time at 10 °C (Table 3.8.2). Some branchedchain alcohols (3-methylbutan-2-ol and 3-methylhexan-2-ol) and pentan-1-ol occurred only in
the headspace of milk spoiled with P. fragi at different times The only distinctive aldehyde was
nonanal, found in the headspace of P. fragi and P. rhodesiae after 10 days and at the end of
storage, respectively. Among distinctive ketones, 3-hydroxybutan-2-one (acetoin) was the most
interesting ketone, due to its absence in UHT control sample and its early detection in milk
spoiled with S. marcescens and P. rhodesiae. Hexan-2-one was detected only in the headspace
of milk samples spiked with P. fluorescens and P. mosselii at the end of storage while P.
rhodesiae was the only species releasing pentane-2,3-dione since 10th day of incubation. The
samples spoiled by P. mosselii and P. fluorescens displayed the production of distinctive
sulphur compounds (methylsulfanyldisulfanylmethane, methylsulfonylsulfanylmethane, 1sulfanylpropan-2-one), being P. mosselii the faster producer (already after 5 days of storage).
Milk contaminated with. S. marcescens was characterized by the presence of pentanoic and 3methylbutanoic acids already after 5 days of storage and heptanoic acid from 10 th day of
incubation while it took P. rhodesiae more than 10 days to produce detectable amount of these
compounds. P. mosselii was found to release the highest diversity of methyl esters during
storage, producing methyl butanoate from the 5th day, and S-methyl butanethioate and S-methyl
ethanethioate after 10 days. The milk contaminated with S. marcescens was the richest in ethyl
esters displaying ethyl butanoate from the 5th day and ethyl hexanoate and ethyl acetate after 10
and 20 days of incubation respectively; in addition, 3-methylbutyl acetate was detected after 10
days of storage. The sample spoiled with P. fragi showed the presence of ethyl acetate, methyl
butanoate and ethyl butanoate after 5, 10 and 20 days respectively. Among hydrocarbons, milk
contaminated with P. fragi was characterized by the presence of 3,3-dimethylhexane during the
entire storage time while heptane and 3-methylhexane were produced only by P. rhodesiae after
10 days of storage as well as hexane in milk spoiled with P. fluorescens.
Overall, among alcohols, 3-methylbutan-1-ol and 2-methylpropan-1-ol were the predominant
alcohols in the headspace of spoiled milk samples during storage time at 10°C, being P. fragi
the species that produced the greater diversity of alcohols. P. rhodesiae resulted as the species
producing the highest number and diversity of ketones (9 different ketones) during storage at
10°C, whereas Morales et al. (2005) found that strains belonging to P. rhodesiae and P.
lundensis produced seven different ketones in cheese samples, being P. lundendis the strongest
producer. P. mosselii was by far the strongest sulphur compound-producing species (7 different
sulphur compounds). Our results are in agreement with those of Morales et al. (2005) where
methylsulfanylmethane was the major sulphur compound being P. libanensis, P. brenneri and
P. rhodesiae the strongest producers. P. rhodesiae was the species producing the highest
number of fatty acids, in agree with results reported for the headspace of cheese made from
milk samples inoculated with six different Pseudomonas strains, where P. rhodesiae produced
high amounts of butanoic and hexanoic acids and the highest levels of total acids (Morales et
al., 2005). The highest production of fatty acids was noticed also in milk spoiled with S.
marcescens, responsible of flavour defects and impairment in milk and dairy products during
cold storage for its ability to produce lipase as reported by Abdou (2003). Moreover, Serratia
genus has been already described in milk (Tornadijo et al., 1993) and cheese (Martin-Platero et
al., 2009) and has been shown to affect milk and cheese sensory quality due to high proteolytic
activities and dimethyl sulphide production (Morales et al. 2003; Chaves-Lopez et al., 2006;).
P. fragi resulted the strongest producer of ethyl-acetate responsible for the fruity flavor defect,
as previously reported in literature (Pereira & Morgan, 1958; Reddy et al., 1968; Cormier et al.,
1991; Hayes et al., 2002). Other esters were detected in milk spoiled with P. mosselii and S.
marcescens, differently from Morales et al.(2005) that found P. rhodesiae the strongest esterproducing species in cheese samples.
52
Table 3.8.2 VOCs detection in spoiled UHT milk during storage time (days) at 10°C
5
10
20
P. fluorescens
PS14
5
10 20
a
nd
nd
nd
nd
nd
nd
b
t
nd
nd
t
nd
t
nd
nd
+
nd
t
nd
nd
nd
nd
+
nd
nd
nd
nd
++
nd
nd
nd
Aldehydes
Exanal
Heptanal
Octanal
Nonanal
Benzaldehyde
+
+
nd
nd
+
+
+
nd
nd
+
+
+
t
nd
+
+
+
t
nd
+
+
+
t
nd
+
ketones
Acetone
Butan-2-one
3-Hydroxybutan-2-one
Pentan-2-one
Hexan-2-one
Heptan-2-one
6-Methylhept-5-en-2-one
Nonan-2-one
Butane-2,3-dione
Pentane-2,3-dione
++
++
nd
+
nd
+
+
t
nd
nd
++
++
nd
+
nd
+
+
+
nd
nd
++
++
nd
++
nd
+
+
+
+
nd
++
+
nd
+
nd
+
+
+
nd
nd
++
+
nd
+
nd
+
+
+
+
nd
VOCs
Alcohols
2-Methylpropan-1-ol
3-Methylbutan-1-ol
3-Methylbutan-2-ol
3-Methylhexan-2-ol
Pentan-1-ol
UHT control
P. fragi PS55
5
P. rhodesiae
PS62
5
10 20
P. mosselii
PS39
5
10 20
S. marcescens
S92
5
10 20
10
20
+
+
+
nd
+
+
+
+
+
+
+
+
+
+
+
+
++
nd
nd
nd
+
++
nd
nd
nd
+
++
nd
nd
nd
nd
+
nd
nd
nd
+
+
nd
nd
nd
+
++
nd
nd
nd
+
++
nd
nd
nd
+
++
nd
nd
nd
+
++
nd
nd
nd
+
+
t
nd
+
+
+
t
nd
+
+
+
t
+
+
+
+
t
t
+
+
+
t
nd
+
+
+
t
nd
+
+
+
t
+
+
+
+
t
nd
+
+
+
t
nd
+
+
+
t
nd
+
+
+
t
nd
+
+
+
t
nd
+
+
+
t
nd
+
++
++
+
++
+
+
+
+
+
nd
++
+
nd
+
nd
+
+
t
nd
nd
++
+
nd
+
nd
+
+
+
nd
nd
++
+
nd
+
nd
+
+
+
+
nd
++
++
+
+
nd
+
+
+
++
nd
++
++
+
+
nd
++
+
+
++
+
++
++
++
++
nd
++
+
+
++
+
++
+
nd
+
nd
+
+
t
+
nd
++
++
nd
++
nd
+
+
+
+
nd
++
++
nd
++
+
+
+
+
+
nd
++
t
+
+
nd
+
+
+
++
nd
++
t
++
++
nd
++
+
+
++
nd
++
+
++
++
nd
++
+
+
+
nd
53
5
10
20
P. fluorescens
PS14
5
10 20
Sulphur compounds
Methylsulfanylmethane
Methyldisulfanylmethane
Methylsulfanyldisulfanylmethane
Methylsulfonylmethane
Methylsulfonylsulfanylmethane
Methylsulfinylmethane
1-Sulfanylpropan-2-one
t
+
nd
+
nd
nd
nd
t
+
nd
+
nd
nd
nd
+
++
nd
+
nd
nd
nd
t
+
nd
+
nd
nd
nd
++
+
nd
+
nd
+
+
Fatty acids
Acetic acid
Butanoic acid
Pentanoic acid
Hexanoic acid
Heptanoic acid
Octanoic acid
3-Methylbutanoic acid
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
+
nd
+
nd
nd
nd
Esters
Ethyl hexanoate
Ethyl butanoate
3-Methylbutyl acetate
Ethyl acetate
S-Methyl ethanethioate
S-Methyl butanethioate
Methyl butanoate
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
VOCs
UHT control
5
10
20
P. rhodesiae
PS62
5
10 20
++
+
+
+
+
+
+
+
+
nd
+
nd
nd
nd
++
+
nd
+
nd
nd
nd
++
+
nd
+
nd
+
nd
++
+
nd
+
nd
+
nd
++
+
nd
+
nd
+
nd
+
+
nd
+
nd
+
nd
+
++
+
+
+
t
+
+
++
+
+
+
+
+
++
++
+
+
+
+
+
++
+
nd
+
nd
+
nd
++
+
nd
+
nd
+
nd
++
+
nd
+
nd
+
nd
nd
+
nd
+
nd
+
nd
+
+
nd
+
nd
+
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
+
+
nd
+
nd
+
nd
+
++
nd
++
nd
+
nd
++
++
+
++
nd
++
nd
++
++
+
++
+
++
+
nd
+
nd
nd
nd
nd
nd
nd
++
nd
nd
nd
nd
nd
t
++
nd
+
nd
+
nd
++
+
+
+
nd
nd
+
++
+
+
+
+
+
+
++
+
+
+
+
+
+
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
+
nd
nd
nd
nd
nd
nd
+
nd
nd
+
nd
+
nd
+
nd
nd
+
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
+
nd
nd
nd
nd
+
+
+
nd
nd
nd
nd
+
+
+
nd
+
nd
nd
nd
nd
nd
+
+
+
nd
nd
nd
nd
+
+
+
+
nd
nd
nd
P. fragi PS55
P. mosselii
PS39
5
10 20
S. marcescens
S92
5
10 20
(Continued)
54
VOCs
UHT control
5
10 20
Hydrocarbons
Heptane
nd nd nd
3-Methylhexane
nd nd nd
3,3-Dimethylhexane
nd nd nd
Hexane
nd nd nd
a
nd, not detected
b
t, molecole occuring in trace amount, < 4 logArea
+, relative abundance between 6,5 – 5 logArea
++, relative abundance between 7 -8 logArea
P. fluorescens
PS14
5
10 20
5
10
20
P. rhodesiae
PS62
5
10 20
nd
nd
nd
nd
nd
nd
+
nd
nd
nd
+
nd
nd
nd
+
nd
nd
nd
nd
nd
nd
nd
nd
+
P. fragi PS55
nd
nd
nd
+
55
+
+
nd
nd
+
+
nd
nd
P. mosselii
PS39
5
10 20
S. marcescens
S92
5
10 20
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
Results and discussion: Characterization of VOCs in milk contaminated with psychrotrophs
Regarding to VOCs releasing after 20 days storage at 5 ˚C (Table 3.8.3) a common feature in
GC profiles both of UHT control milk and all contaminated samples was the presence of the
2,3,3-trimethyl pentane hydrocarbon. It is worthwhile to note that this compound was not
produced by psychrotrophic strains during storage at 10°C. Differently some ketones (nonan-2one, butane-2,3-dione) and sulphur compounds (methyldisulfanylmethane and
methylsulfinylmethane) were not observed in UHT control milk and in samples spoiled with P.
fluorescens, P. fragi and P. mosselii at 5 °C differently from the same samples incubated at 10
˚C. 2-methylpropan-1-ol alcohol was produced only by P. rhodesiae. At 5 ˚C no detection of
fatty acids was recorded in milk samples spoiled with P. fragi and P. mosselii. P. rhodesiae
produced only acetic acid while the highest number of fatty acids was found in milk spoiled
with P. fluorescens and S. marcescens.
Table 3.8.3 VOCs detected in spoiled UHT milk at 5°C after 20 days of storage
VOCs
Alcohols
2-methylpropan-1-ol
3-methylbutan-1-ol
UHT
control
T0
T20
a
P.
fluorescens
PS14
P.
fragi
PS 55
P.
rhodesiae
PS62
P.
mosselii
PS39
S.
marcescens
S92
nd
nd
nd
nd
nd
+
nd
+
+
+
nd
+
nd
+
Aldehydes
Exanal
Heptanal
Octanal
Benzaldehyde
+
+
b
t
+
+
+
t
+
+
+
t
+
+
+
t
+
+
+
t
+
+
+
t
+
+
+
t
+
ketones
Acetone
Butan-2-one
Pentan-2-one
Heptan-2-one
6-methylhept-5-en-2-one
Nonan-2-one
Butane-2,3-dione
++
++
+
+
t
t
t
++
++
+
+
+
t
t
++
+
+
+
+
nd
+
++
+
+
+
+
+
t
++
+
+
+
+
+
+
++
+
+
+
+
+
+
++
+
+
+
+
+
+
Sulphur compounds
Methylsulfanylmethane
Methyldisulfanylmethane
Methylsulfonylmethane
Methylsulfinylmethane
nd
+
+
nd
t
+
+
nd
+
nd
+
t
+
+
+
t
+
+
+
+
+
+
+
nd
+
+
+
+
Fatty acids
Acetic acid
Butanoic acid
Hexanoic acid
Octanoic acid
nd
nd
nd
nd
nd
nd
nd
nd
+
+
+
nd
nd
nd
nd
nd
+
nd
nd
nd
nd
nd
nd
nd
nd
+
+
+
+
+
+
Hydrocarbons
2,3,3-trimethyl pentane
+
+
+
+
a
nd, not detected; bt, molecole occuring in trace amount, < 4 logArea
+, relative abundance between 6,5 – 5 logArea
++, relative abundance between 7 -8 logArea
56
Results and discussion: Characterization of VOCs in milk contaminated with psychrotrophs
By comparing the chromatographic VOC profiles of spoiled milk samples at different stages of
storage (both at 10°C and 5°C), it was possible to identify compounds that could be regarded as
potential markers of psychrotrophic contamination.
The 3-methylbutan-1-ol branched-chain alcohol could be considered as a possible marker of
psychrotrophic contamination due to its absence in unspoiled UHT milk and its production in
all spoiled milk samples already from 5th day of storage at 10° and after 20 days at 5°C. 3methylbutan-1-ol could derive from the proteolytic activity of the strains and from leucine
catabolism (Smit et al., 2005). Moreover, among the detected volatiles, some molecules, absent
or found in minimal traces in UHT control milk, have already been detected in spoiled samples
after 5 days of storage at 10°C, so they could be regarded as a markers of bacterial spoilage
development: 2-methylpropan-1-ol (P. fragi, P. rhodesiae, S. marcescens), 3-hydroxybutan-2one (acetoin) (P. rhodesiae and S. marcescens), butanoic (P. fluorescens, P. rhodesiae, P.
mosselii, S. marcescens) and hexanoic acids (P. fluorescens, P. rhodesiae, S marcescens),
butane-2,3-dione (P. rhodesiae, P. mosselii and S. marcescens).
Some metabolites were detectable only when the milk was contaminated by a specific
psychrotrophic strain during storage at 10°C. 3-methylbutan-2-ol, 3-methylhexan-2-ol and
pentan-1-ol and 3,3-dimethylhexane hydrocarbon were only produced by P. fragi; pentane-2,3dione, heptane and 3-methylhexane were detectable only in milk contaminated with P.
rhodesiae while hexane was produced only by P. fluorescens.
There are several possible origins for detected bacterial volatile metabolites and the range of
organic compounds that can be used as carbon and energy source by psychrotrophic bacteria is
wide. Although it is difficult, and in some cases not possible, to attribute them to a specific
biosynthetic pathway, some hypotheses can be made. Most of the compounds identified in this
work could be associated with the microbial and enzymatic activities occurring during the
spoilage of milk. It is known that aldehydes, resulted from the catabolism of amino acid,
(Marilley & Casey, 2012) can be reduced to alcohols by alcohol dehydrogenases (e.g. 3methylbutanal to 3-methyl-1-butanol) or oxidized to carboxylic acids by an aldehyde
dehydrogenase (e.g. 3-methylbutanal to isovaleric acid/3-methylbutanoic acid). Additionally,
pyruvate or citrate are starting materials for the formation of short-chain flavor compounds such
as 3 hydroxybutan-2-one (acetoin) and butane-2,3-dione through glycolytic and lactate
converting fermentations (Michal, 1999) The catabolism of pyruvate and leucine seems to play
an important role in case of P. rhodesiae and S. marcescens since the products of these
metabolic pathway (3-methyl-1-butanol, 2,3-butanedione, 3 hydroxybutan-2-one) were
significantly found in the headspace of milk spoiled with these bacteria. Sulfur compounds
usually arise from sulforated amino acids catabolism (Montel, 1998; Ardo, 2006). Some methyl
ketones such as nonan-2-one can derive from a lypolytic process but also from several other
possible pathways, such as alkane degradation (Stanyer et al., 1996; Padda et al., 2001)
bacterial dehydrogenation of secondary alcohols (Lukins & Foster, 1963; Padda et al., 2001).
Ethyl esters were formed through chemical esterification of alcohols and carboxylic acids
following microbial esterase activity (Talon et al., 1998; Toldra, 1998). Carboxylic acids
deriving from hydrolysis of triglycerides and phospholipids (Toldra, 1998) can also be an initial
substrate for oxidation with the formation of final odorous products. Aldehydes, such as
hexanal, nonanal and also alcohols can derive from hydrolysis of triglycerides or from amino
acid degradation (Montel, 1998).
3.8.2 Sensory analysis of microbially spoiled milk samples
The individual ability of each of the five psychrotrophic strains to produce odor changes in
UHT milk was tested after 10 days at 10°C, in order to determine which volatile organic
57
Results and discussion: Characterization of VOCs in milk contaminated with psychrotrophs
compounds are associated to undesirable odor characteristics. Different sensory evolution
patterns were observed according to the strains. Sensory characteristics were slightly better in
the sample spoiled with P. fluorescens, while P. fragi was classified as moderately spoiling
micro-organism. The poorest sensory qualities were found in samples contaminated with P.
rhodesiae, P. mosselii and S. marcescens, considered the strongest odour spoiling microorganisms with different evolutions in their spoilage activity. In milk contaminated with P.
fluorescens “fresh milk” and “dairy odor” off-flavours were detected by panelists. P. fragi
exhibited mainly “cottage cheese”, “sweet whipped cream”, “slightly fruity” and “strong butter”
off-odors while for P. mosselii sulphur and rotten off-odours were noted. For the milk spoiled
with P. rhodesiae a sensory descriptor of “baby vomit” and acid odours were systematically
suggested by panelists and adverse sensory effects have been also detected in milk spoiled with
S. marcescens, producing metallic/oxidized and grassy odours. Our results showed that the
spoilage potential of the five psychrotrophic strain was revealed by their ability to produce
different off odours, most likely induced by volatile compounds. Volatile compounds detected
in milk sample spoiled by P. mosselii could be responsible for characteristic flavors, such as
sulphur compounds for rutten/putrid odors while 3-hydroxybutan-2-one, 2,3-butanedione, 3methyl-1-butanol and an high number of fatty acids produced by P. rhodesiae and S.
marcescens led to the formation of strong butter/buttermilk and sour, rancid and nauseous offodors. P. fragi displayed a moderate spoilage potential by producing typical fruity off-odors due
to the release of esters, as was already reported in several studies (Cormier et al., 1991) P.
fluorescens milk samples were always described as adequate for consumption considering the
off-flavour as a minor sensory change, probably due to a less production of distinctive VOCs
during storage time. This finding is in accordance with the results of sensory analysis of the
spoilage aromas of milk containing Pseudomonas species by Hayes et al. (2002).
Sensory analysis confirmed the differences in the bacteria-specific VOC profiles found by
SPME/GC-MS analysis that contributed to the overall decrease in milk quality.
In the visual aspect, the descriptive analysis detected slight structural changes, such as
coagulation, for milk samples inoculated with P. fluorescens and P. fragi whereas P. rhodesiae,
P. mosselii and S. marcescens resulted the faster milk-coagulating strains.
58
Results and discussion: Characterization of VOCs in milk contaminated with psychrotrophs
3.9 Conclusions
It may be concluded from the results obtained in the present work that psychrotrophic strains
belonging to different species of the genus Pseudomonas and Serratia marcescens produce a
large variety of VOCs in milk during storage time and that these abilities are species-dependent,
as reported by Morales et al. (2005) regarding the VOCs production in cheese by Pseudomonas.
A general increase in the magnitude of the peaks of volatile compounds was observed with
increasing temperature and storage time confirming that VOC profile of psychrotrophic strains
is time-temperature dependent. In addition, the presence of these bacteria affect negatively the
structural and sensory characteristics of milk, being P. rhodesiae, P. mosselii and S. marcescens
the main spoilage species. Using SPME/GC-MS method, it was possible to identify potential
markers for psychrotrophic milk spoilage (3-methylbutan-1-ol, 2-methylpropan-1-ol, 3hydroxybutan-2-one, butane-2,3-dione, butanoic and hexanoic acids).
Determination of milk off-flavours has been the subject of numerous investigations (Badings &
Neeter, 1980; Urbach, 1990; Kim & Morr, 1996; Contarini & Povolo, 2002; Toso et al., 2002;
Marsili, 2003; Karatapanis et al., 2006) but SPME/GC-MS analysis of contaminated milk with
psychrotrophic bacteria, mainly Pseudomonas, is a relatively new field of research and still
requires extensive basic research to evaluate the candidate compounds, which may serve as
biomarkers.
In seeking potential markers of milk spoilage, we feel that the emphasis should be placed on
screening for headspace compounds or fragments produced by more strains and species of
psychrotrophs in milk. In the future, it would be of utmost interest to study in deep the VOC
presence in spoiled milk, confirming the possible markers detected in this work and finding new
ones for prevention of quality loss and extension of milk shelf-life.
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63
Results and discussion: Fatty acids released from cream by psychrotrophs
3.11 Fatty acids released from cream by psychrotrophs isolated from bovine raw milk
Use of cooling, imperfect cleaning and inadequate disinfection of milking machines in primary
milk production remain the main reasons for the occurrence of psychrotrophic bacteria in raw
milk. On average, the psychrotrophic microbiota of bulk raw milk accounts between 10 and
50% of the total bacterial load (Chambers, 2005). Also psychrotrophic bacteria are mesophile
organisms, they are able to grow at low temperatures and, moreover, they are endowed with
proteolytic and lipolytic activities (Cousin, 1982). Munsch-Alatossava and Alatossava (2006)
considered this group to be the most relevant spoiling microorganisms in the dairy industry
because of their capacity to produce heat resistant enzymes (proteinasas and lipases) responsible
of quality issues and sensory defects, resulting in a limited shelf-life of milk and dairy products.
The triglyceride fat fraction of milk accounted by more than 96% of total lipid, located in the
core of fat globule, which are arranged from a conjugated ester of long chained fatty acids with
glycerol molecules, enveloped by a thin layer composed phospholipids, glycolipids, and
several proteins, (Evers et al., 2008). Obviously, to release the free fatty acids (FFAs) and cause
changes and flavour defects (rancid or soap flavours if, respectively, short chain or medium or
long chain fatty acid are released) in the milk, several consecutive cleavages are necessary,
beginning with the breaking of the surrounding globule fat membrane followed by the lysis of
the triglycerides into glycerol and fatty acids, which are accumulated in the milk gave rise to the
above mentioned defects (Stepaniak 2004). The fat globule is susceptible to the action of many
lipolytic agents, such as physiological, physiomechanical, and chemical factors (Deeth & FitzGerald, 2006), the latter involving lipases (McPherson & Kitchen, 1983). Lipases are
carboxylesterases that hydrolyse the bond ester of acylglycerols Those that hydrolyse
acylglycerols of less than 10 carbon-chain fatty acids are named esterases, or carboxylases (EC
3.1.1.1) while those hydrolysing acylglycerols more than 10 carbon-chain fatty acids are the
lipases, or triacylglycerol acylhydrolases (EC 3.1.1.3). Esterases are active in aqueous solutions,
while true lipases work in lipid–water interfaces rather than in aqueous phase (the ‘‘interfacial
activation phenomenon’’) (Brockerhoff & Jensen, 1974; Anthonsen et al., 1995) and most are
also capable of hydrolysing esterase substrates (Jaeger et al., 1994; 1999). Lipolytic enzymes
can be of endogen origin, in which case most of the lipases are associated with the casein
micelle structure (Stepaniak, 2004) or of exogenous origin, secreted as a result of the bacterial
metabolic action.
It is generally known that Pseudomonas spp. produce thermostable lipases (McPhee &
Griffiths, 2011) active on milk fat and responsible for spoilage as a result of the production of
short and medium chain fatty acids (Deeth, 2006). A high FFA accumulation causes the
deterioration of technological properties (e.g. a worse whipping ability of cream) and sensory
attributes of milk, which is reflected by a rancid or soap off-flavour that may negatively
influence the quality of dairy products (Antonelli et al., 2002, Hanuš et al., 2008). According to
Vyletělová et al. (2000) the medium-chain fatty acids (C12–C16) are primarily liberated by the
action of Pseudomonas fluorescens lipolytic enzymes while short-chain fatty acids (C6–C10)
are sporadically released or in very small quantities.
Despite many researches dealing with the ability of psychrotrophs, mainly Pseudomonas spp.,
to produce thermostable enzymes and their effect on the most important compounds of milk,
fatty acids profiles of lipolytic psychrotrophs belonging to different genera and species has not
been well fully explained. Due to the relevance of the aforementioned issues, the present work
was envisaged in an attempt to clear up the ability of psychrotrophic bacteria, belonging to
different bacterial species, to break lipids and to quantify the fatty acids released as a result of
this action.
64
Results and discussion: Fatty acids released from cream by psychrotrophs
3.12 Material and methods
3.12.1 Microorganisms
The strains tested for the lipid breakdown in this study were previously isolated from raw milk
samples, identified and characterized for their enzyme activity (Decimo et al., 2014). Fifteen
psychrotrophic strains were selected showing different lipolytic activity at 7 and 22°C:
Pseudomonas fluorescens (n=5) P. fulva (n=2), P. putida (n=1), Ps. mosselii (n=1), P. fragi
(n=1), P. rhodesiae (n=1), Hafnia alvei (n=1) and Serratia marcescens (n=3).
3.12.2 Physico-chemical analysis of cream
For experiments, UHT cream purchased in the market was used. The pH of cream was
determined using a Crison Digit-501 pH-meter (Crison Instruments LTD, Barcelona, Spain).
Water activity (aw) was measured using a Decagon CX1 hygrometer (Decagon Devices Inc.,
Pullman, WA, USA) at 25 °C. Moisture content (oven air-drying method) and ash (muffle
furnace) were analysed following the AOAC (1995) method. The fat content was determined
using the method of Bligh and Dyer described by Hanson and Olley (1963). All determinations
were performed in triplicate and mean values of the replicates were considered.
3.12.3 Preparation of partially purified extracellular lipase
The selected strains was subcultured in Brain Heart Infusion (BHI) broth (Biolife, Milan, Italy)
supplemented with 2% of skim milk and incubated at 30°C for 24 h. Then, one mL of each
culture was inoculated in 500 mL erlenmeyer sterile flasks containing 250 mL of the above
mentioned culture medium BHI. The flasks were incubated at 30°C with constant shaking at
125 rpm for 48 h. to stimulate enzyme production. After incubation, the culture was centrifuged
at 12,000 g for 30 min at 4°C to separate cells and, then, the supernatant was precipitated with
ammonium sulphate (NH4)2SO4 to 50% saturation and stirred for 2 h on ice bath. After
centrifugation at 12,000 g for 30 min at 4°C, the pellet was recovered by dissolving it in TrisHCl 0,1 M pH 7.4 buffer and, afterwards, dialyzed overnight against the same buffer at 4°C and
then collected in a sterile vial. The final solution was lyophilized (Telstar, Lioalfa-10, Madrid,
Spain) and the dry extract stored by frizzing at - 20°C. For analysis, the lyophilizated was
dissolved in a minimal volume of Tris-HCl 0,1 M pH 7.4.
3.12.4 Changes in the substrate specificity of crude enzyme
The specificity of the lipase activity of partially purified lipase was studied using four different
triacylglycerides (TAGs), namely, glyceryl esters of tributyrate, tricaproate, trimyristirate and
trioleate. The substrate media were prepared by sonicating 1%, 0.1%, 0.06% and 0.06% of the
above mentioned triacylglycerides, respectively 100 mL of Agar Noble (Difco, BD, Sparks,
Md.) for 2 min using an ultrasonic apparatus (model UP-400S, Dr. Hielscher GmbH, Stuttgart,
Germany), which operates at 24 kHz, 400 W and 60 µm. The medium with triacylglycerols was
plated in Petri dishes and 50 µl of each of the 15 crude enzymes was deposited into the wells
(diameter 6 mm). The specific lipase activity was defined by a clear or turbid zone formation
around the wells after incubation at 30°C. The halo performed was periodically recorded with a
calliper from 24 h of incubation until its stabilization. The area was measuring using the
following formula: A = π [ (r + r')2 - r'2] being r the radius of the halo and r' the radius of the
well (3mm).
65
Results and discussion: Fatty acids released from cream by psychrotrophs
3.12.5 Preparation of spoiled cream samples
One mL of each partially purified lipase of the 15 psychrotrophic strains was used to inoculate
20 mL of UHT cream and the mixture incubated at 30°C for 24 h, according to Ouattara et al.
(2004) who reported that a significant lipolysis is observed in the first 12-24 h. The incubation
was extented for four additional days to verify the changes in the free fatty liberated with the
incubation time. After that, the cream samples were lyophilized and frozen until gas
chromatrography analysis.
3.12.6 Free fatty acid analysis
For quantitative lipid extraction the method proposed by Segura and Lopez-Bote (2014) was
applied. Briefly, in a 2 mL safe-lock micro test tube, 170 mg of lyophilised spoiled cream
sample were accurately weighed. Two steel balls (2 mm diameter) and 1.5 mL of
chloroform/methanol (8/2) (v/v) mixture were added. After being tightly capped, the tubes were
placed on the adapters and homogenized for 2 min at 20 Hz in a Mixer Mill MM400 (Retsch
Technology, Haan, Germany). The final biphasic system was allowed to separate by
centrifugation (8 min, 12,000 g). The solvent was decanted into a previously weighed 4 mL
vial. The extraction was repeated three times. Solvent was evaporated under nitrogen stream at
25 °C. Samples were kept in a desiccator until constant weight (about 2 h). Lipid content was
then gravimetrically determined.
The lipid fractionation into major lipid classes (apolar lipids , AL, free fatty acids, FFA and
polar lipids, PL) was carried out according to the method of Ruiz et al. (2004). Briefly, a 0.2 g
lipid extract was dissolved in a mixture of hexane/chloroform/methanol (95/3/2) (v/v/v) in the
proportion 4/3 (w/v) and added to a 500 mg aminopropyl minicolumn (Varian, Harbor City,
USA), which was previously activated with 7.5 mL of hexane. AL and FFA were eluted with 5
mL of chloroform and diethyl ether/acetic acid (98/2) (v/v), respectively. PL were successively
eluted with 2.5 mL methanol/chloroform (6/1) (v/v) and 2.5mL of 0.05 M sodium acetate in
methanol/chloroform (6/1) (v/v). Both fractions were combined and analysed together.
Total lipid extracts were methylated by dissolving the sample in 1 mL of 5% sodium methylate
methanolic solution in culture tubes (Olivares et al., 2009). After being tightly capped, the tubes
were vortexed and heated for 1 h at 70 °C, shaking every 15 min. After tempering, 1 mL of 5%
sulphuric acid methanolic solution was slowly added. The tubes were vortexed and shaking
again in the previously conditions. After tempering, the fatty acids methyl esters (FAMEs) were
extracted two times with 1.5 mL of petroleum ether and directly injected in the GC system.
Identification and quantification of FAMEs were performed by gas–liquid chromatography
following the conditions described by Segura and Lopez-Bote (2014), modifying the oven
temperature. The FAMEs were analyzed in a gas chromatograph (HP 6890 Series GC System)
equipped with flame ionization detector and a J&W GC Column, HP-Innowax Polyethylene
Glycol (30 m × 0.316 mm × 0.25 µm). Nitrogen was used as a carrier gas. The oven
temperature was programmed for initial holding for 1.50 min at 80 °C, then heating to 210 °C at
a rate of 4.5 °C/min , followed by a second heating to 250 °C at a rate of 7 °C/min followed by
a final holding for 3 min. The flame ionization was held at 250 °C. FAMEs peaks were
identified by comparing their retention times with those of the authentic internal standard,
(tridecanoic acid methyl ester) from Dr. Ehrenstorfer GmbH, Augsburg, Germany.
66
Results and discussion: Fatty acids released from cream by psychrotrophs
3.13 Results and discussion
3.13.1 Physico-chemical characteristics of cream
Selected physico-chemical parameters (moisture, fat content, ash, aw and pH) of UHT cream
were determinated, achieving the following values (percentage on wet matter): moisture 59.44 ±
0.03; fat 11.34 ± 3.62; ash 0.41 ± 0.01; aw 0.949 ±0.006 and pH 6.77 ± 0.05.
3.13.2 Lipolytic activity of partially purified lipases
Lipases from Pseudomonas spp. can actively hydrolyze a variety of natural vegetable oils and
synthetic TAGs (emulsified triglyceride substrates) ranging from tributyrin to triolein
(Stepaniak & Sorhaug, 1989; McPhee & Griffiths, 2011), and they show preference for
triglycerides rather than monoglycerides, and for medium-chain glycerides, such as those
containing 8 to 19 carbon fatty acids (Bozoglu et al., 1984). P. fluorescens lipase showed the
highest lipolytic activity with regard to long chain TAGs, especially triolein. Most of the lipases
have a preference for the sn-1 and sn-3 positions of TAGs (Rogalska et al., 1993). In this study
the lipolytic activity from the fifteen selected psychrotrophic strains on the TAGs
aforementioned in material and methods was investigated.
The changes in lipolytic activity on tributyrin, tricaproin, trimyristin and triolein of
psychrotrophic strains are shown in figures 3.13.1, 2, 3 and 4, respectively. Generally, all
strains presented lipolytic activity against the selected triacylglycerols and as expected, the
activity of most strains increased with the incubation time. The lipolytic strains more active
toward tributyrin were P. fragi PS55 and P. fulva PS1 while P. rhodesiae PS62 and S.
marcescens S92 showed the lowest activity. The others psychrotrophic lipases showed a
variable activity on this acylglycerol. The halo area at 216 h of incubation ranged between 6.16
cm2 for P. fragi PS55 and 1.99 cm2 for P. rhodesiae PS62 (Figure. 3.13.1). The lipolysis
patterns observed on other substrates were quite similar to that of the tributyrin even the
absolute levels achieved after 216 hours Briefly, in tricaproin (Figure. 3.13.2), the halos were
comprised between 6.3 cm2 of P. fluorescens PS14 and 2.09 cm2 of P. mosselii PS39. In the
case of trimyristin (Figure. 3.13.3) two strains (P. rhodesiae PS62 and P. mosselii PS39) gave
rise to the lowest halos area (1.45 and 1.21 cm 2, respectively). On the contrast, the halos of five
strains (P. putida PS17, P. fragi PS55, P. fluorescens PS73, P. fluorescens PS81 and P.
fluorescens PS56) reached the highest final values, between 5.15 and 4.37 cm2, i.e. 3.5 to 4-fold
higher than those of P. rhodesiae PS62 and P. mosselii PS39. The remaining strains showed
intermediate activities. Finally, for triolein (Figure 3.13.4), four strains (P. fluorescens PS14, P.
putida PS17, P. fluorescens PS81 and P. fluorescens PS73) showed a clear higher lipolytic
power (halos area between 5.2 and 3.9 cm2), than the remaining ones, which gave rise to halos
lower than 3.0 cm2. It is noteworthy that P. fluorescens PS81strain showed the fastest activity
since evident at 24 h of incubation while the time to detect the lipase activity elapsed, in
general, 48 – 72 hours when tricaproin and trimyristin were used as substrate and more than 72
hours in the case of triolein. These results partially agree with those of Schuepp et al. (1997)
that reported that the FII from P. fragi CRDA037 presented the highest activity on trimyristin
followed on triacetin and then on tributyrin. Nishio et al. (1987) reported that the purified
extracellular lipase from P. fragi 22.39B exhibited the highest affinity toward tributyrin, in
agreement with the data here obtained. P. fluorescens PS14 displayed high activity on both
glycerol triocaproate and trioleate, which is in partial agreement with Chakraborty and Paulraj
(2009) that indicated a preferential specificity of the lipase from P. fluorescens MTCC 2421
toward cleaving shorter short-chain length fatty acid TAGs but a scarce hydrolytic action
against toward monounsaturated TAGs (i.e. triolein) with longer fatty acyl chain length. P.
67
Results and discussion: Fatty acids released from cream by psychrotrophs
putida PS17 showed high specificity toward the fatty acid triglycerides having medium to long
carbon chain lengths (C14:0- C18:1n9) while P. fulva PS1 exhibited higher affinity on
tributyrin P. rhodesiae PS62 was the strain with the lowest lipolytic activity against all tasted
TAGs.
68
Results and discussion: Fatty acids released from cream by psychrotrophs
Tributyrin (C4:0)
7
P. mosselii PS39
P. rhodesiae PS62
6
P. fluorescens PS74
P. putida PS17
2
Area halo (cm )
5
P. fluorescens PS56
4
P. fluorescens PS14
S. marcescens S92
3
P. fragi PS55
S. marcescens S33
2
P. fluorescens PS73
P. fluorescens PS81
1
H. alvei PS57
0
P. fulva PS10
0
24
48
72
96
120
144
168
192
216
P. fluorescens PS23
P. fulva PS1
Time (h)
Figure 3.13.1 Specificity of lipases from psychrotrophic strains toward tributyrin TAG
Tricaproin (C8:0)
7
P. mosselii PS39
P. rhodesiae PS62
6
P. fluorescens PS74
P. putida PS17
P. fluorescens PS56
P. fluorescens PS14
2
Area halo (cm )
5
4
S. marcescens S92
P. fragi PS55
S. marcescens S33
3
P. fluorescens PS73
P. fluorescens PS81
2
H. alvei PS57
P. fulva PS10
1
P. fluorescens PS23
P. fulva PS1
0
0
24
48
72
96
120
144
168
192
216
Time (h)
Figure 3.13.2 Specificity of lipases from psychrotrophic strains toward tricaproin TAG
69
Results and discussion: Fatty acids released from cream by psychrotrophs
Trimyristin (C14:0)
6
P. mosselii PS39
P. rhodesiae PS62
5
2
Area halo (cm )
P. fluorescens PS74
P. putida PS17
4
P. fluorescens PS56
P. fluorescens PS14
3
S. marcescens S92
P. fragi PS55
S. marcescens S33
2
P. fluorescens PS73
P. fluorescens PS81
1
H. alvei PS57
P. fulva PS10
0
P. fluorescens PS23
0
24
48
72
96
120
144
168
192
216
P. fulva PS1
Time (h)
Figure 3.13.3 Specificity of lipases from psychrotrophic strains toward trimyristin TAG
Triolein (C18:1n9)
6
P. mosselii PS39
P. rhodesiae PS62
5
P. fluorescens PS74
P. putida PS17
P. fluorescens PS56
P. fluorescens PS14
2
Area halo (cm )
4
S. marcescens S92
3
P. fragi PS55
S. marcescens S33
P. fluorescens PS73
2
P. fluorescens PS81
H. alvei PS57
P. fulva PS10
1
P. fluorescens PS23
P. fulva PS1
0
0
24
48
72
96
120
144
168
192
216
Time (h)
Figure 3.13.4 Specificity of lipases from psychrotrophic strains toward triolein TAG
70
Results and discussion: Fatty acids released from cream by psychrotrophs
3.13.3 Release of FFAs by psychrotrophs
To confirm the qualitative analyses of lipolytic activity, a study on the release of fatty acids
from cream was performed by use of gas liquid chromatography. The results are presented in
figure 3.13.5, in which the free fatty acids values reached after 1 and 4 days are shown. In
control samples was observed a minor increase of FFAs, which has been attributed to the effect
of the both the natural lipoprotein lipase (LPL) and the growth of the background microbiota
(Taylor & MacGibbon, 2003). In inoculated samples, it may be observed that the lipolytic
activity of the strains was variable. Briefly, among the fifteen tested lipolytic psychrotrophic
strains, the species producing the highest concentrations in total FFAs at 24 h of incubation
were P. fluorescens PS73 and S. marcescens S33 but the former was one of the most active
after four days while S. marcescens S33 hardly increased the FFAs concentration in this period.
The strongest total fatty acids liberated after 4 days of incubation were due to the P. fluorescens
Total FFAs (mg/g tot lipids)
160
140
120
100
24 h
80
4 days
60
40
20
0
Figure 3.13.5 Total FFAs concentration (mg/g total lipid) in control and inoculated samples
after incubation at 30 °C for 24 h and 4 days
PS81 activity. On the opposite side, in spite of P. putida PS17 was one of the major FFAs
producer at 24 h, it did not show a significant increase in total FFAs concentration after 4 days
of incubation, which could be related with the metabolic adaptation or reduction in the
availability to break the milk fat fraction during the time. Other strains had an intermediate
behavior, which may be clear deduced in figure 3.13.5.
71
Results and discussion: Fatty acids released from cream by psychrotrophs
In order to analyze the individual fatty acids liberated, four strains were selected, as model of
high (P. fluorescens PS73), intermediated (S. marcescens S92), odd behavior (initially low
activity but it accelerates during storage) (P. fragi PS55) and low (P. mosselii PS39) activity.
Eighteen FFAs (C-8:0 to C-20:1) were identified in both the control cream lipid extract and
inoculated samples, the FFAs detected in the highest quantities were, in general, those of the
intermediate par carbon chain (C-12:0 to C-18:0) and unsaturated C-18:1 and C-18:2. This fact
coincided with the most abundant triacylglycerols in the milk fat (Jensen, 1992). Other authors
have reported similar phenomenon, e.g. Outtara et al. (2004) found that milk sample inoculated
with P. fragi CBS-15-5260 the C-16:0 was the fatty acid detected in the largest amount (16.3
mg/kg) followed by C-18:1 (12.8 mg/kg), C-18 (9.8 mg/kg) and C-14 (7.3 mg/kg) although in
this work the dominant FFA was C-18:1 (see table 1). The increased of the mentioned FFAs
during cream storage was quite regular for P. fluorescens PS73 and S. marcescens S92 although
the percentage of increased were variable (table 1) between 200 to 350% and 100 to 200%,
respectively. Both strains roughly represent the action of the majority strains studied in the
present research. On the other hand, the trend observed for P. fragi PS55 was different. This
was the one of the lowest lipolytic strain after 24 hours, but very high increases of FFAs were
observed during storage since the values were 4 to 7-fold higher after four days. This means
that the average increase percentage was about 350% although in many fatty acid the
percentage was higher than 450% (table 3.13.1) such as C-10:0, C-12:0, C-16:1 and others. The
P. mosselii PS39 was also a strain with an initial low activity and the increase in the FFAs
during storage was negligible.
These results indicated that the ability of lipolytic psychrotrophic strains to release fatty acids
from milk fat varies according to the bacterial genus, among species and even with the strain of
given specie. The results of the present work are in total agreement with those of Ouattara et al.
(2004), who stated that lipolytic activity of psychrotrophic bacteria common in raw milk was
species-specific. Therefore, no general prognosis of the concentration of FFA released could be
made although it may be predicted that, in a greater or lesser degree, a lipid breakdown will
occur if the raw milk storage is prolonged.
The proteolytic phenomenon are probably the major event of concern occurred in milk and
dairy products but the fatty acid release by hydrolysis of milk triacylglycerols through the
activity of psychrotrophic lipases are the main source primary cause for the undesirable changes
in the flavour of these products. The normal content of FFA in milk is very low, accounting for
a 0.1% of total fat (Walstra and Jenness, 1984) but if this concentration increases in about a 1%,
it may be result in a rancid flavor in milk, which is caused by the accumulation of short-chain
FFAs (butyric, caproic and caprylic acids). However, a higher amount of medium-chain FFAs
(capric, lauric and myristic acids) leads to a soapy taint while long-chain saturated fatty acids,
namely palmitic and stearic acids contribute little to off-flavour (Al-Shabibi et al., 1964;
Vulfson, 1994). As psychrotrophic bacteria are able to develop under refrigeration, it is
convenient to minimize its growth on raw milk and cream in order to avoid a high bacteria
number during storage and, along with, to diminish the lipolytic activity. As is well known,
these bacteria do not survive even mild heat treatment. It should be sufficient a thermization
process (about 65 ºC for few seconds) to reduce Pseudomonas spp (and other Gram negative
psychrotrophs) to a large extent. This reduction should be enough to produce negligible changes
in both milk nutrients and sensory properties.
72
Results and discussion: Fatty acids released from cream by psychrotrophs
Table 3.13.1 Concentrations of free fatty acids (mg/100 g total lipid) in control and inoculated
samples after incubation at 30°C for 1 and 4 days by four selected psychrotrophic strains
FFA
P. fragi PS55
1
day
4 days
C8:0
1.29
C10:0
C12:0
P. fluorescens PS73
Δ
1
day
4
days
a
1.78
+
0.44
0.40
2.15
++++
0.46
3.06
++++
C14:0
1.40
7.59
C14:1
0.14
1.06
C15:0
0.17
C15:1
0.04
C16:0
4.81
C16:1
0.25
C17:0
0.17
C17:1
0.06
C18:0
2.06
8.12
C18:1
5.20
28.11
C18:2
0.47
3.22
C18:3
0.04
0.46
C18:4
0.17
0.40
C20:0
ND
C20:1
0.33
Total
17.46
S. marcescens S92
Δ
1
day
4
days
1.27
++
0.53
0.89
3.16
+++
1.24
4.71
+++
++++
3.05
11.58
++++
0.42
1.63
0.72
+++
0.31
0.15
+++
0.07
18.97
+++
1.64
++++
0.35
0.30
P. mosselii PS39
Δ
1
day
4
days
0.84
+
1.01
1.12
+
0.72
1.75
++
0.69
0.93
+
0.99
2.82
++
0.85
0.95
+
+++
2.60
7.05
++
2.75
2.75
-
+++
0.33
1.03
+++
0.24
0.25
+
1.08
+++
0.27
0.67
++
0.29
0.29
-
0.22
+++
0.06
0.14
++
0.07
0.07
-
8.72
28.11
+++
7.79
18.01
++
8.97
8.98
-
0.66
2.31
+++
0.62
1.64
++
0.56
0.56
-
++
0.16
0.50
+++
0.14
0.33
++
0.17
0.22
+
++++
0.13
0.45
+++
0.11
0.31
++
0.09
0.11
+
+++
4.10
11.65
++
3.65
7.84
++
3.81
3.82
-
++++
12.11
41.05
+++
9.26
29.15
+++
6.37
7.23
+
++++
1.49
4.21
++
1.16
3.00
++
0.72
0.97
+
++++
0.19
0.74
+++
0.14
0.51
+++
0.08
0.11
+
++
0.20
0.81
+++
0.15
0.53
+++
0.10
0.11
+
0.18
-
0.06
0.16
++
0.05
0.17
+++
0.05
0.05
-
0.54
+
0.22
0.69
+++
0.17
0.50
++
0.15
0.16
-
78.80
++++
34.44
114.33
28.73
76.30
26.98
28.30
+
a
+++
a
++
a
Δ
a
Δ (1→4) - Percentage of the lipolytic activity increase after four days of the cream storage: + (< 100 %);
++ (100 - 200 %); +++ (200 - 350 %); ++++ (> 350 %)
3.14 Conclusions
Lipolysis in milk and dairy products derived from psychrotrophic bacteria is a constant concern
in the dairy industry due to the production of off-flavours and off-odours. In this study, the
lipolytic activity of fifteen psychrotrophic strains isolated from bovine raw milk has been
assayed. Although it seems that the lipolytic activity of psychrotrophs is a general
characteristic, no general pattern for acylglycerols breakdown was found since the behavior of
selected strains was variable, being S. marcescens S33 and P. fluorescens PS81 the major FFAs
producers, at 24 h and 4 days, respectively.
Moreover lipases from psychrotrophic strains showed a variable range of specificity toward
fatty acid esters with different fatty acid chain lengths, being P. fragi PS55, P. putida PS17, P.
fluorescens PS14 and P. fulva PS 10 the more active to cleave fatty acid triglycerides.
A thorough understanding of the mechanism of lipolysis and of the ability to control FFAs
release by psychrotrophic bacteria during storage of milk may be required to minimize lipaserelated problems, to avoid the deterioration of sensory properties and maintain the overall
quality of dairy products.
73
Results and discussion: Fatty acids released from cream by psychrotrophs
3.15 References
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295–296.
Anthonsen HW. Lipases and esterases: A review of their sequences, structure and evolution. In
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Antonelli ML et al., 2002. Determination of free fatty acids and lipase activity in milk: quality
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AOAC. 1995, Official methods of analysis (16th ed.). Washington, DC, USA, Association of
Official Analytical Chemists.
Bozoglu F. et al., 1984, Isolation and characterization of an extracellular heat-stable lipase
produced by Pseudomonas fluorescens MC50. J Agric Food Chem 32:2–6.
Brockerhoff H, Jensen RG. Lipolytic enzymes. In VII. Milk lipases, Academic Press, New
York, USA 1974, pp. 118–129.
Chakraborty K, Paulraj R, 2009, Purification and biochemical characterization of an
extracellular lipase from Pseudomonas fluorescens MTCC 2421. J Agric Food Chem 57:38593866.
Chambers JV. The Microbiology of raw milk. In Dairy microbiology handbook: The
microbiology of milk and milk products, Robinson RK ed., John Wiley & Sons, Inc., Hoboken,
NJ, USA 2005, pp. 39–90.
Cousin MA. 1981, Presence and activity of psychrotrophic microorganisms in milk and dairy
products: a review. J Food Prot 45:172–207.
Deeth HC 2006, Lipoprotein lipase and lipolysis in milk. Int Dairy J 16:555–562.
Deeth HC, Fitz-Gerald CH. Lipolytic enzymes and hydrolytic rancidity. In Advanced dairy
chemistry volume 2: lipids, Fox PF, McSweeney PLH, eds., 3rd ed., Springer , New York,
2006, pp 1–76.
Evers JM et al., 2008, Heterogeneity of milk fat globule membrane structure and composition
as observed using fluorescence microscopy techniques. Int Dairy J 18:1081–9.
Hanson SWF, Olley J, 1963, Application of the Bligh and Dyer method of lipid extraction to
tissue homogenates. Biochem J 89:101-102.
Hanuš O et al., 2008, Analysis of raw cow milk quality according to free fatty acid contents in
the Czech Republic. Czech J Anim Sci 53:17–30.
Jaeger KE et al., 1994, Bacterial lipases. FEMS Microbiol Rev 15:29–63.
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Jaeger KE et al., 1999, Bacterial biocatalysts: Molecular biology, three-dimensional structures,
and biotechnological applications of lipases. Annu Rev Microbiol 53:315–351.
Jensen RG. Fatty acid in milk and dairy products. In Fatty acid in foods and their health
implications, Chow CK, ed., Marcel Dekker, Inc. New York, 1992, pp. 95-135.
McPherson AV, Kitchen BJ, 1983, Reviews of the progress dairy science: the bovine milk fat
globule membrane – its formation, composition, structure and behavior in milk and dairy
products. J Dairy Res 50:107–33.
McPhee JD, Griffiths MW. Pseudomonas spp. In Encyclopedia of Dairy Sciences, Vol. 4,
Roginski H, Fuquay WJ, Fox FP, eds., Academic Press 2002 , pp. 2340-2350.
McPhee JD, Griffiths MW. Pseudomonas spp. Elsevier Ltd, Guelph University, Guelph, ON,
Canada, 2011.
Munsch-Alatossava P, Alatossava T, 2006, Phenotypic characterization of raw milk-associated
psychrotrophic bacteria. Microbiol Res 161:334–6.
Nishio T et al., 1987, Purification and some properties of lipase produced by Pseudomonas
fragi 22.39 B. Agric Biol Chem 51:181–186.
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concentration on intramuscular fat content and fatty acid composition in pigs. Meat Sci 82:6–
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Outtara GC et al., 2004, Fatty acids released from milk fat by lipoprotein lipase and lipolytic
psychrotrophs. J Food.Sci 69:659-663.
Rogalska E et al., 1993, Stereoselective hydrolysis of triglycerides by animal and microbial
lipases. Chirality 5:24–30.
Ruiz J et al., 2004, Improvement of a solid phase extraction method for analysis of lipid
fractions in muscle foods. Anal Chim Acta 520:201–205.
Schuepp C et al., 1997, Production, partial purification and characterization of lipases from
Pseudomonas fragi CRDA 037. Process Biochem 32:225–232.
Segura J, Lopez-Bote CJ, 2014, A laboratory efficient method for intramuscular fat analysis.
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Stepaniak L, Sørhaug T. Biochemical classification. In Enzymes of psychrotrophs in raw food,
McKellar RC ed., CRC Press, Boca Raton, Florida 1989, pp. 35-55.
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Vyletelova M et al., 2000, Effects of lipolytic enzymes Pseudomonas fluorescens on liberation
of fatty acids from milk fat. Czech J Food Sci 18:175-182.
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76
Results and discussion: Proteomic characterization of extracellular proteases
3.16 Proteomic characterization of extracellular proteases of P. fluorescens strains isolated
from raw milk
The microbiota of raw milk depends on the health status of the cattle, the type of feed and the
storage conditions. It has been reported that intensive cleaning of milking equipment together
with prolonged refrigerated storage of raw milk at farm and processing plants favour the growth
of psychrotrophic bacteria, particularly Pseudomonas spp. (MacPhee & Griffiths, 2002; Lafarge
et al., 2004). The Pseudomonas genus corresponds to a diverse and ecologically significant
group of bacteria which are found in a very large number of natural environments (terrestrial,
freshwater or marine). Some of them are also plant, animal and human pathogen or responsible
for food spoilage (Palleroni, 1992; Gonzalez et al., 2000; Park et al., 2000). Psychrotrophic
bacteria are the key microorganisms responsible for spoilage of milk and dairy products, due to
their ability to produce heat-resistant proteases that hydrolyse casein, leading to formation of
bitter off-flavours and gelation of UHT milk (Mitchell & Marshall, 1989; Sorhaug & Stepaniak,
1997; Chen et al., 2003; Datta & Deeth, 2003;). Production of extracellular protease by
Pseudomonas strains is a common feature during the late log phase and in the stationary phase
of bacterial growth (Hellio et al., 1993; Rajmohan et al., 2002). The production of at least one
monomeric extracellular protease of molecular weight varying between 23 kDa and 56 kDa,
appears to be a common feature among strains of P. fluorescens, which has been reported as the
predominant species among psychrotrophic bacteria (Ahn et al., 1999; Dogan & Boor, 2003;
Dufour et al., 2008). Most research has focused on expression, production and biochemical
properties of proteases produced by P. fluorescens strains associated with spoilage of milk dairy
products (Barach et al., 1976; McKellar, 1989; Mitchell & Marshall, 1989; Kohlmann et al.,
1991; Ching-hsing & McCallus, 1998; Liao & McCallus, 1998; Koka & Weimer, 2000;
Nicodème et al., 2005; Liu et al., 2007; Dufour et al., 2008). Most of these proteases are
metalloproteases which are rich in alanine and glycine residues and poor in cysteine and
methionine residues. Calcium is essential for the activity and stability of these proteases
(Mitchell et al., 1986). In milk, they preferentially hydrolyze the caseins κ > β> αS1. One
metalloprotease called AprX which belongs to the serralysin family, has been characterized in
P. fluorescens and its gene (aprX) was identified in several strains (Martins et al., 2005;
Marchand et al., 2009). The aprX gene products of two P. fluorescens strains revealed
estimated molecular weights of 50 kDa (Ching-hsing & McCallus, 1998) and 48.9 kDa
(Kumura et al., 1999). Similar molecular weights were reported by other researchers,
investigating food spoiling proteases (Koka & Weimer, 2000; Rajmohan et al., 2002; Nicodème
et al., 2005, Marchand et al., 2009).
The aim of this work was to evaluate the extacellular caseinolytic potential of three P.
fluorescens strains (PS19, PS60 and PS24), which have previously been isolated from bovine
raw milk (Decimo et al., 2014). Moreover, proteolytic enzyme of PS19 Pseudomonas
fluorescens strain, which showed the highest caseinolytic activity, was subjected to proteomic
analysis.
3.17 Material and methods
3.17.1 Bacterial strains and culture conditions
In a previous work (Decimo et al., 2014), a large set of proteolytic psychrotrophic strains was
isolated from bovine raw milk samples. Strains were further identified by a polyphasic
(phenotypic and molecular) approach and tested for their proteolytic activity by agar diffusion
assays. From this collection, three P. fluorescens strains (PS19, PS60 and PS24), which showed
the highest proteolytic enzyme production on milk agar, were selected and further investigated.
77
Results and discussion: Proteomic characterization of extracellular proteases
The production of proteases by these P. fluorescens strains was examined in minimal salt
medium (MSM) containing K2HPO4 (0.7%); KH2PO4 (0.2%), MgSO4·7H2O (0.02%),
(NH4)2SO4 (0.1%), glycerol (0.4%) supplemented with CaCl 2 at a final concentration of 1 mM
(MS medium; Liao & McCallus, 1998). Growth was at 22 °C under stirring (180 rev min -1) and
aerobic conditions.
3.17.2 Preparation of crude enzyme extract
Cryopreserved strains were firstly recovered in Brain Heart Infusion broth (BHI) (Biolife,
Milan, Italy) + 1 mM CaCl2, before inoculation in MSM medium and incubated at 22 °C until
growth was visually present (approx. 6 hours). Next, 300 µl (3%) of incubated BHI + 1 mM
CaCl2 broth was inoculated in 10 ml of MSM medium + 1 mM CaCl2 and incubated overnight
at 22 °C. This was repeated once again to stimulate the adaptation of the strains to the minimal
salt medium with 1 mM CaCl2 (Marchand et. al, 2009). Then, 30 ml of incubated MSM + 1 mM
CaCl2 was inoculated 2 l Erlenmeyer flasks containing 1L of MSM +1 mM CaCl 2. After 24 h of
incubation at 22 °C under stirring (125 rev min -1) and aeration, cells were harvested by
centrifugation (6 000 g for 20 min at 4 °C) The filter-sterilized (0.22 µm) supernatant was
heated at 95 °C for 8.45 min (Marchand et al., 2008, 2009) to select for heat-resistant proteases,
and used as crude enzyme extract after storing at -20 °C before casein zymography.
3.17.3 Casein zymography and detection of extracellular caseinolytic activity
To detect the extracellular caseinolytic activity of the three P. fluorescens strains and to
estimate the molecular mass of their bacterial proteases, a casein zymography was performed.
After heating, 16 µl of each cell free supernatants (CFS), obtained from growth of the P.
fluorescens strains in MSM, was dissolved in a 156.25 mM Tris-HCl buffer, pH 6.8 in the
presence of 10% (w/v) SDS, 62.5% glycerol and 0.5% (w/v) bromophenol blue (5x Loading
Buffer). Twenty µl of CFS solution were loaded for electrophoresis on a 12% polyacrylamide
gel polymerized with 0.1% (w/v) casein (Sigma–Aldrich, Milan, Italy) (sodium caseinate) under
non-reducing conditions, i.e. without β-mercaptoethanol. The samples were run initially at 60 V
for 30 min and at 100 V for 100 min. The molecular mass standard ranged from 10kDa to 250
kDa (PageRuler Plus Prestained protein Ladder, Thermo Scientific, Milan, Italy). After
electrophoresis migration, the gel was washed in 2.5% (v/v) Triton X-100 (renaturing solution)
for 30 min and then incubated overnight at 37 °C in development buffer (50 mM Tris, 200 mM
NaCl, 5 mM CaCl2×2H2O and 0.02% (v/v) Brij 35 (Sigma), pH 7.5). After incubation the gel
was stained with 0.1% (w/v) Coomassie Brilliant Blue R-250 dissolved in a mixture of 50%
methanol (v/v) and 10% acetic acid (v/v) (staining solution), followed by discoloration in a
solution containing 50% methanol (v/v) and 10% acetic acid (v/v) (destaining solution) until
proteolytic activity appeared as clear bands on a blue background.
P. fluorescens PS19 crude enzyme extract was concentrated in a stirred cell ultrafiltration unit
(Centricon-30; Millipore (UK) Ltd, Watford, UK) with a 10 kDa membrane and further tested
on zymogram gel with 0.1% (w/v) of single casein fractions (κ, β, αS1, Sigma).
3.17.4 Proteomic analysis of crude enzyme extract from PS19 P. fluorescens strain
P. fluorescens PS19 proteolytic bands of approximately 15, 25 and 45 kDa (as determined by
zymogram analysis) was subjected to proteomic characterization. Proteomic analysis was
performed using a LTQ-Orbitrap system (working in nanoLC–ESI-MS/MS mode), using a
bottom-up approach, which involved denaturation, reduction, alkylation of Cys residues prior to
enzymatic digestion of the protein and ESI-MS analysis of digestate. In detail, proteases were
78
Results and discussion: Proteomic characterization of extracellular proteases
digested in-gel solution and in-solution solution (FASP, filter aided sample preparation), using
trypsin (Roche Diagnostics, Monza, Italy) as digestion enzyme.
3.17.5 In gel digestion
After destaining, gel lanes were excised and cut into three slices each corresponding to
migration regions of different molecular weights (15, 25 and 45 kDa). Gel slices were
subsequently digested with trypsin according to a protocol previously described by Lamond Lab
(2007).
Briefly, gel pieces were incubated for 60 min at 56 °C with 10 mM dithiothreitol. (DTT) in 50
mM NH4HCO3 for cysteine reduction, immediately followed by incubation with iodoacetamide
(IAA) 55 mM in 50 Mm NH4HCO3 for 45 min at room temperature and in the dark, for
cysteines alkylation. After discarding iodoacetamide, gel pieces were rinsed several times in 50
mM NH4 HCO3 (digestion buffer) and finally incubated in 100% acetonitrile (ACN) for
dehydration. About one µg of trypsin (Roche) dissolved in 50 mM NH4HCO3 was incubated
with each gel slice. After overnight digestion at 37 °C, the tryptic mixture was acidified with 2
µl of 50% trifluoroacetic acid solution and collected. Gel pieces were incubated for 10 minutes
with 30% acetonitrile/3% trifluoroacetic acid (TFA) (extraction solution); the solution was
collected and pooled with the initial peptide mixture. Gel pieces were incubated for additional
10 minutes with 100% acetonitrile; the solution was pooled with the initial one. The digestates
were evaporated on a vacuum concentrator to about 5 µl. The peptides present in the
concentrates were solubilized in 100 µl of 0.1% formic acid, desalted and concentrated using
reverse-phase C18 handmade nano-columns (StageTips) (Rappsilber et al., 2007). Samples
loaded on C18 StageTips were eluted with 80% acetonitrile, lyophilized and re-suspended in 10
µl of 0.1% formic acid for LC–MS/MS analysis.
3.17.6 In solution digestion
The digestion protocol reported by Manza et al. (2005) and Wisniewski et al. (2009) was also
applied. This protein in-solution digestion was carried out using spin filter devices (filter-aided
sample preparation (FASP) protocol).
The concentrated crude enzyme extract was transferred to a Microcon spin filter device
(Millipore Amicon Ultra MWCO 3000 Da) (max 500 µl) and mixed with 250 µl of Urea buffer
(UA) containing 8 M urea in 0.1 M Tris-HCl, pH 8.5. Filter units are used to remove interfering
chemicals and small molecules and mass spectrometry-incompatible substances, before
digestion, without substantial sample loss. The device was centrifuged at 14,000 × g for 20 min
and the eluate was thrown away. All subsequent centrifugation steps were performed under the
same conditions, allowing maximum concentration. Subsequently, 250 µl of DTT 10 Mm in
UA (reducing solution) was added to the concentrated protein mixture followed by
centrifugation. Then incubation with 250 µl of IAA 55mM in UA (alkylating solution) was
performed for 30 min at room temperature in the dark. The device was centrifuged to remove
excess iodoacetamide solution. Two additional wash steps were performed by adding 400 µl of
UA buffer followed by centrifugation. About 2 µg of LysC (Roche) dissolved in 50 mM
NH4 HCO3 was incubated with protein mixture for 3 h.
The concentrated protein mixture was also subjected to trypsin digestion by adding 1 µg in 50
mM NH4 HCO3 before diluiting urea concentration (< 1M) with 200 µl of 50 mM ammonium
bicarbonate. Trypsin digestion was performed by incubation overnight at room temperature.
Afterward, peptides were collected in a low-binding tube using centrifugation, and the filter
device was rinsed with 100 µl of ammonium bicarbonate and again centrifugated at 14,000 g for
10 min to recover all peptides. Eluted peptides were desalted and concentrated using reverse-
79
Results and discussion: Proteomic characterization of extracellular proteases
phase C18 handmade nano-columns (StageTips) (Rappsilber et al., 2007) and evaporated on a
vacuum concentrator to about 5 µl. Samples loaded on C18 StageTips (The concentrated
peptides) were stocked at -20 °C and re-suspended in 15 µl of 0.1% formic acid for nanoLC–
MS/MS analysis.
3.17.7 NanoLC–ESI-MS/MS peptide analysis
Chromatographic separation was performed by injecting 5 µl of digested peptides resuspended
in 0.1% formic acid on a C18 HALO PicoFrit column (75 µm × 10 cm, 2.7 µm particle size,
100Å, New Objective, USA). The sample was injected by an Ultimate3000 RSLCnano system
and electrosprayed using a nanoESI source (Thermo). Peptide separation was performed by a
reverse phase linear gradient from 1% acetonitrile, 0.1% formic acid to 40% acetonitrile, 0.1%
formic acid over 60 min, at flow rate of 300 nl/min. The instrument operated in data-dependent
mode to acquire both full MS and MS/MS spectra. Full MS spectra were acquired in profile
mode by the FT analyzer in a scan range equal to 300–1500 m/z, using capillary temperature
220 °C, AGC scan 5 × 105 and resolution 60,000 FWHM at m/z 400. Tandem mass spectra
were acquired by the linear ion trap (LTQ) for the 2 most intense ions exceeding 1 × 10 4 counts.
MS/MS spectra acquisition was set as follows: centroid mode, resolution 15,000, precursor ions
isolation width of 2 m/z, AGC target 1 × 10 4 and normalized collision energy 30 eV. Dynamic
exclusion was enabled to reduce redundant spectra acquisition as follows: 3 repeat counts, 30 s
repeat duration, 45 s of exclusion duration. The mass spectrometer and spectra analysis were
fully automated and controlled by the Xcalibur software (version 2.0.7,Thermo Scientific) and
operated in data-dependent acquisition (DDA) mode, to automatically switch between MS and
MS/MS scans.
3.17.8 Mass spectral data elaboration and database searching
The software Proteome Discoverer 1.4 (Thermo) was used to extract peaks from spectra and to
match them against a database dealing with Pseudomonas microorganisms (updated
08.07.2014.)
The selected protease was trypsin (cleaving at the C-terminus of Lys and Arg, unless followed
by Pro), with maximum 2 allowed missed cleavages. One static (carbamidomethylation on Cys)
and 2 dynamic modifications (deamidation on Asn and oxidation on Met) were selected. The
precursor mass tolerance was set to 5 ppm, and fragment mass tolerance was set to 0.5 Da.
Low-confidence peptide matches were filtered out by selecting peptide confidence = high. For
each protein identified, a minimum of at least two unique matching peptides was considered as
a prerequisite for protein validation with a high degree of confidence.
3.18 Results and discussion
3.18.1 Extracellular caseinolytic potential of P. fluorescens PS19, PS60 and PS24
Among the psychrotrophic strains isolated from bovine raw milk in a previous work (Decimo et
al, 2014), three strains (PS19, PS60 and PS24) exhibited different proteolytic activity at 7 °C
and 22 °C. These P. fluorescens strains, after growth in MSM supplemented with 1 mM CaCl 2,
showed proteolytic activity on milk agar-well plates at 22 °C. Growth and enzyme production
were examined in MSM containing 1 mM CaCl2. because this medium was reported to increase
extracellular protease production in Ps. fluorescens (Amrute & Corpe, 1978). Moreover, Liao
and McCallus (1998) indicated that the divalent cation Ca 2+ is essential for the activity of the
aprX enzyme and they used this medium for the biochemical and genetic characterization of an
80
Results and discussion: Proteomic characterization of extracellular proteases
extracellular protease from Ps. fluorescens CY091. Rajmohan et al. (2002) and Nicodeme et al.
(2005) also found that the addition of CaCl2 to the growth medium significantly enhanced the
specific-proteolytic activity of Pseudomonas spp.
The heat-resistant caseinolytic activity of the cell free supernatant of each P. fluorescens strains
was further evaluated by casein zymography and one heat-stable protease of approximately 45
kDa was detected for all the three strains (Figure 3.18.1).This result is in agreement with data
reported by other researchers (Noreau & Drapeau, 1979; Kohlmann et al., 1991; Ching-hsing &
McCallus, 1998; Koka and Weimer, 2000; Nicodème et al., 2005; Dufour et al., 2008,
Marchand et al., 2009). All tested strains produced protease at 22 °C, but the intensity of the
protease patterns on zymogram varied; P. fluorescens PS19 exhibited the highest extracellular
caseinolytic activity while strains PS60 and PS24 displayed a weak extracellular proteolytic
potential. This might be indicative for a variable expression and/or activity of the proteases.
Besides, this variability in regulation might be temperature dependent (McKellar & Cholette,
1987; Burger et al., 2000; Nicodème et al., 2005, Marchand et al., 2009). P. fluorescens PS19,
being the more proteolytic strain, was further investigated and the extracellular caseinolytic
activity of its concentrated (ultrafiltered at 10 kDa) protease extract was examined on
zymogram with sodium caseinate and single casein fractions (αs, β and κ). Surprisingly, two
additional proteolytic bands with molecular masses of approximately 15 and 25 kDa was
visualized on zymography gels (Figure 3.18.2). The presence of proteolytic activity due to low
molecular weight bands was also reported by Rajmohan et al. (2002) after ultrafiltration of the
culture supernatans of a P. fluorescens strain. The appearance of these bands was attributed to
the concentrating effect of the applied ultrafiltration process before zymogram analysis
(Rajmohan et al., 2002). Nevertheless, Marchand et al. (2009) reported that same Pseudomonas
strains, after 8 days of incubation in milk, displayed 2 or 3 clearance zones of lower molecular
weight on casein zymography. They assumed the appearance to be likely attributable to
substrate depletion, by which the protease, being the only protein source left, is degraded into
smaller active fragments. Although the production of low molecular mass proteases by a single
strain of P. fluorescens has been reported (Rajmohan et al., 2002), the identification of the
putative 15 and 25 kDa proteases has never been described. For this reason, the crude
proteolytic extract of P. fluorescens PS19 was subjected to proteomic characterization.
81
Results and discussion: Proteomic characterization of extracellular proteases
1
2
3
4
Figure 3.18.1 Extracellular caseinolytic activity of
P. fluorescens strains: casein zymography under
non reducing conditions.
1. Protein molecular weight ladder, 6.5–200 kDa
2. PS19 proteolytic enzyme extract
3. PS24 proteolytic enzyme extract
4. PS60 proteolytic enzyme extract
55
45
55
55
35
35
25
15
25
15
A)
B)
55
55
35
35
25
25
15
15
C)
D)
Figure 3.18.2. Extracellular caseinolytic activity of P. fluorescens strain PS19: casein
zymography under non reducing conditions of concentrated proteolytic enzyme extract with A)
0,1% of sodium caseinate; B) 0,1% of αs – casein; C) 0,1% of β – casein and D) 0,1% of κ –
casein.
1. Protein molecular weight ladder, 10–250 kDa
2 and 3. PS19 concentrated proteolytic enzyme extract
82
Results and discussion: Proteomic characterization of extracellular proteases
3.18.2 NanoLC–ESI-MS/MS and identification of P. fluorescens PS19 extracellular
proteases
The proteases of P. fluorescens PS19, as visualized at 45, 25, and 15 kDa on the zymogram
(Fig. 2), were analyzed by nLC/MS/MS analysis after both in gel and in solution trypsinolysis.
The aim was to verify if they correspond to any known protein of Pseudomonas spp. Nanoscale
liquid chromatography coupled to tandem mass spectrometry (nanoLC–MS/MS) has become an
essential tool in the field of proteomics for the analysis of peptides derived by enzymatic
digestion of either purified proteins or moderately complex protein mixtures. In fact, its
sensitivity has advantages over conventional LC–MS/MS that allow the analysis of peptide
mixtures in sample-limited situations (Gaspari & Cuda, 2011).
Although MS strategies for the protein identification may vary (depending on the type of
modification and on the available MS instrumentation), the analysis of either intact proteins
(top–down approach) and/or peptides generated from enzymatic digestion (bottom–up or
peptide mapping approach) is the most widely used approach (Reid & McLuckey, 2002).
For our purpose, we focused on bottom-up approach by digesting proteases the P. fluorescens
PS19 strain both in gel and in solution.
The nLC/MS/MS analysis of the in gel trypsin-digested band at 45 kDa excised from the
Coomassie-stained gel showed that it was similar to the sequence of P. fluorescens AprX
metalloprotease C9WKP6 (SwissProt, accessed 20.02.2014) with a high coverage (55.14%).
(Figure 3.18.3).
The nLC/MS/MS analysis of the in gel trypsin-digested protein band with molecular mass of 15
kDa excised from the SDS-PAA gel showed similarity to both the sequences of another AprX
metalloprotease (accession number: E6Z7L2; coverage: 8.36%) and Protein F (accession
number: Q51777; coverage: 10.73%) from P. fluorescens.
Only one matching peptide (MEPSSGLELIRK) was found for the in gel trypsin digestion of the
25 kDa band excised from the gel. It showed an homology to the sequence of hypothetical
protein of P. fluorescens (accession number: CAA72727) and response regulator of
Pseudomonas sp. TKP (accession number: WP_024077763).
tr|C9WKP6|C9WKP6_PSEFL
MNETHASSPKAEPLSAARAVAAGIDFAELQVGPHGLFWNEYRPEDAACRV
-------MSKVKDKAIVSAAQASTAYSQIDSFSHLYDR-----------.*.: : . *. *. ::::: .*
tr|C9WKP6|C9WKP6_PSEFL
WQWRDGQAHCLTPPTFSVRSRVYEYGGGAFCLTDDGLVFVNEADQQLYRQ
--------------------------GGNLTVNGKPSYTVDQAATQLLRD
** : :...
*::* ** *:
tr|C9WKP6|C9WKP6_PSEFL
SLSDETPEVLTSSECRYGDLQFANGQVLAVEENRDRHRLVAINLAGGQRH
GAAYRDFDGNGKIDLTY-------------------TFLTSATQSTMNKH
. : . :
. : *
*.: . : ::*
tr|C9WKP6|C9WKP6_PSEFL
LLAEGADFYAAPTLSPDGRRLAWIEWNRPDQPWTATRLMVAERQRDHTFT
GISGFSQFNTQQKAQAALAMQSWADVAN------VTFTEKASGGDGHMTF
:: ::* : . ..
:* : .
.*
*.
.*
tr|C9WKP6|C9WKP6_PSEFL
QPRCVAGDGAQESLQQPRFDDSGRLYCLTDRGGFWQPWVESTEGLSPLPS
GNYSSGQDGAAAFAYLP----------GTGAGYDGTSWYLTNNSYTPNKT
. . ***
*
*. *
.* :.:. :* :
tr|C9WKP6|C9WKP6_PSEFL
AAADHGPAPWQLGGCTWLPLSEDTYLASWTEEGFGRLGLCHGDGSREDFT
PDLNN------------YGRQTLTHEIGHTLGLAHPGDYNAGNGNPTYND
. ::
. *: . *
.
*:*.
83
Results and discussion: Proteomic characterization of extracellular proteases
tr|C9WKP6|C9WKP6_PSEFL
GDYSRFRHLAQDDQFIYCIAASPVSSASVIAIERKSRQVKTLAGGVVPLP
ATYGQDTRGYSLMSYWSESNTNQNFSKGGVEAYASGPLIDDIAAIQKLYG
. *.: : . .:
:.
* . :
.. :. :*.
tr|C9WKP6|C9WKP6_PSEFL
ADQISRPQTLRYPSGSGEAHGFFYPAMNDDTKPPLVVFIHGGPTSACYPM
ANFNTRATDTTYGFNSNTGRDFLSATSNAD-KLVFSVWDGGGNDTLDFSG
*: :*.
* .*. .:.*: .: * * * : *: ** : :.
tr|C9WKP6|C9WKP6_PSEFL
FDPRIQYWAQRGFAVADLNYRGSSGYGRAYRQALHLSWGDVDVEDACAVV
FTQ------NQKINLTATSFSDVGGLVGNVSIAKGVTIENAFGGAGNDLI
*
:: : :: .: . .*
* :: :.
. ::
tr|C9WKP6|C9WKP6_PSEFL
GYLAERGLIDGDKAFIRGGSAGGYTALCALAFHKIFRAGASLYGVSDPVA
IGNQVANTIKGGAGNDLIYGGGGADQLWGGAGSDTFVYGASSD--SKPGA
. *.*. .
..**
* . * . * ***
*.* *
tr|C9WKP6|C9WKP6_PSEFL
LGRVTHKFEGDYLDWLIGNPVEDAERYAARTPLLHANNISVPMIFFQGEL
ADKIFDFTSGSDKIDLSG--------------ITKGAGVTFVNAFTGHAG
.:: . .*.
* *
: :. .::.
*
tr|C9WKP6|C9WKP6_PSEFL
DAVVVPQQTRDMVKALQDNGILVEAHYYADERHGFRKAGNQAHALEQEWL
DAVLSYASG----TNLGTLAVDFSGHGVADFLVTTVGQAAASDIVA---***:
.
. *
.: ...* **
. :. :
tr|C9WKP6|C9WKP6_PSEFL
FYRRVME
-------
Figure 3.18.3 ClustalW sequence alignment of protein identified by proteomic analysis of
proteolytic 45 kDa band from P. fluorescens PS19. Peptides as retrivied by mass spectrometry
are shown in bold. Symbols (.), (:) and (*) represent the degree of conservation within the
compared sequences.
The nLC/MS/MS analysis of the in solution trypsin-digest of the concentrated (UF 10 kDa)
proteolytic extract showed that it was similar to the sequences of extracellular metalloprotease
AprX from P. fluorescens (acc. no. C9WKP6; coverage 15.72%), of 60 kDa chaperonin GroEL
from P. fluorescens (acc. no. C0J914; coverage 30.05%; 3) and of 50S ribosomal protein L25
from P. fluorescens (acc. no. F1BCI0; coverage 11.06%).
Combing the results from nLC/MS/MS analysis of both in gel and in solution digestion, the
correspondence of the 45 kDa protease to metalloprotease C9WKP6 was demonstrated. In
addition, it can be likely hypothesized that the 15 kDa protease is a fragment of this AprX
metalloprotease (Fig. 3.18.4). On the contrary, the 25 kDa protease showed no homology to any
known protein of Pseudomonas spp.
84
Results and discussion: Proteomic characterization of extracellular proteases
. :**************:*****
1
MSKVKDKAIVSAAQASTAYSQIDSFSHLYDRGGNLTVNGKPSYTVDQAATQLLRDGAAYR
C9WKP6_PSEFL
1
-------------------------------------SWQPSYTVDQAATQLLREGAAYR
E6Z7L2_PSEFL
** *********************************************************
61
DFDGNGKIDLTYTFLTSATQSTMNKHGISGFSQFNTQQKAQAALAMQSWADVANVTFTEK
C9WKP6_PSEFL
24
DFBGNGKIDLTYTFLTSATQSTMNKHGISGFSQFNTQQKAQAALAMQSWADVANVTFTEK
E6Z7L2_PSEFL
:***********************************************************
121 ASGGDGHMTFGNYSSGQDGAAAFAYLPGTGAGYDGTSWYLTNNSYTPNKTPDLNNYGRQT
C9WKP6_PSEFL
84
TSGGDGHMTFGNYSSGQDGAAAFAYLPGTGAGYDGTSWYLTNNSYTPNKTPDLNNYGRQT
E6Z7L2_PSEFL
************************************************************
181 LTHEIGHTLGLAHPGDYNAGNGNPTYNDATYGQDTRGYSLMSYWSESNTNQNFSKGGVEA
C9WKP6_PSEFL
144 LTHEIGHTLGLAHPGDYNAGNGNPTYNDATYGQDTRGYSLMSYWSESNTNQNFSKGGVEA
E6Z7L2_PSEFL
************************************************************
241 YASGPLIDDIAAIQKLYGANFNTRATDTTYGFNSNTGRDFLSATSNADKLVFSVWDGGGN
C9WKP6_PSEFL
204 YASGPLIDDIAAIQKLYGANFNTRATDTTYGFNSNTGRDFLSATSNADKLVFSVWDGGGN
E6Z7L2_PSEFL
************
301 DTLDFSGFTQNQKINLTATSFSDVGGLVGNVSIAKGVTIENAFGGAGNDLIIGNQVANTI
C9WKP6_PSEFL
264 DTLDFSGFTQNQ-----------------------------------------------E6Z7L2_PSEFL
60
C9WKP6
23
E6Z7L2
120
C9WKP6
83
E6Z7L2
180
C9WKP6
143
E6Z7L2
240
C9WKP6
203
E6Z7L2
300
C9WKP6
263
E6Z7L2
360
C9WKP6
275
E6Z7L2
420
C9WKP6
275
E6Z7L2
361 KGGAGNDLIYGGGGADQLWGGAGSDTFVYGASSDSKPGAADKIFDFTSGSDKIDLSGITK
C9WKP6_PSEFL
276 -----------------------------------------------------------E6Z7L2_PSEFL
421 GAGVTFVNAFTGHAGDAVLSYASGTNLGTLAVDFSGHGVADFLVTTVGQAAASDIVA
C9WKP6_PSEFL
276 --------------------------------------------------------E6Z7L2_PSEFL
477
C9WKP6
275
E6Z7L2
Figure 3.18.4. ClustalW sequence alignment of proteins identified by proteomic analysis of P.
fluorescens PS19 concentrated (UF 10 kDa) proteolytic extract (in solution digest) and
proteolytic band of 15 kDa. Peptide coverage is indicated by highlighted characters: PS19Trypsin retentate (shown in bold), PS19-Trypsin 15 kDa (underlined and shown in bold).
85
Results and discussion: Proteomic characterization of extracellular proteases
3.19 Conclusions
Three strains (PS19, PS24 and PS60) of Pseudomonas fluorescens previously isolated from
bovine raw milk, have been further investigated in this study.
All strains secreted one thermostable protease of about 45 kDa, which was characterized in P.
fluorescens PS19 by nLC/MS-MS and assigned to P. fluorescens AprX metalloprotease
C9WKP6. The proteomic characterization of 15 kDa protease of P. fluorescens PS19 showed a
high similarity with AprX metalloprotease C9WKP6 whereas the 25 kDa protease showed no
homology to any known protein of Pseudomonas spp.
3.20 References
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88
Results and discussion: Hydrolysis of casein fractions by P. fluorescens enzyme extract
3.21 Identification of peptides from hydrolysis of casein fractions by P. fluorescens PS19
enzyme extract
Breakdown of proteins (proteolysis) is important in a wide variety of food products. Proteolysis
may have beneficial effects and may be essential for desirable qualities in food products, such
as flavour development and texture changes during the ripening of cheese. However,
uncontrolled or unwanted proteolysis can adversely affect the quality of foods.
Proteolyis in milk is caused by both native proteases and proteases produced by psychrotrophic
microorganisms during refrigerated storage of the milk (Fairbairn & Law, 1986; Grufferty and
Fox, 1988). Psychrotrophic microorganisms are those that grow at 7 °C, although their optimal
growth temperature may be higher. During cold storage after milk collection, they dominate the
microbiota and are responsible for many quality problems in dairy products. Under sanitary
conditions, <10% of the total microflora are psychrotrophs, compared to >75% under
unsanitary conditions (Suhren, 1989). The numbers of psychrotrophs that develop after milk
collection depend on the storage temperature and time. Among the psychrotrophs,
Pseudomonas spp. are most frequently reported in raw milk. Psychrotrophs produce
extracellular proteases (metalloproteases, that require the presence of a metal ion such as
calcium for optimal activity) (Mitchell and Ewings, 1985) that contribute to sensory defects in
milk and dairy products, releasing (with formation of) peptides, responsible of undesiderable
bitter off-flavour. Optimal enzyme synthesis occurs in the majority of psychrotrophs at 20–30
°C, but considerable synthesis occurs at lower temperatures. For example, production of
extracellular protease by Pseudomonas fluorescens at 5 °C was 55% of that produced at 20 °C
(McKellar, 1982). Heat-stable proteases from psychrotrophs attack all forms of casein, with
preferential hydrolysis for κ-casein, then β-casein, and finally αs-casein (Fairbairn and Law,
1986; Cox, 1993). There is controversy in the literature as to whether the major whey proteins
are susceptible to bacterial proteases (Fairbairn and Law, 1986; Sorhaug and Stepaniak, 1997).
An extracellular protease isolated from P. fluorescens M3/6, produced after incubation in
reconstituted nonfat dry milk stored at 7 °C, was characterized and shown to have activity on α, β-, and κ-caseins (Kohlmann et al., 1991). Hydrolysis of κ-casein can result in destabilization
of the casein micelle and the production of small peptides that contribute to bitter flavours
(Cromie, 1992).
The aim of this work was to characterize by LC/MS the peptidic profiles generated by the
action of thermostable protease from P. fluorescens PS19 strain on milk casein fractions and to
identify casein peptides useful as markers to early recognize the activity of psychrotrophic
strains in (un)processed milk.
3.22 Material and methods
3.22.1 Hydrolysis of casein fractions by enzyme extract from P. fluorescens PS19
The crude enzyme extract was obtained from the P. fluorescens PS19 strain isolated from
bovine raw milk (Decimo et al., 2014), which was selected for its high proteolytic potential on
milk agar plates and extracellular caseinolytic activity on caseinate zymogram gel. The
proteolytic extract from the cell free supernatant of P. fluorescens PS19, grown in minimal salt
(MS) medium supplemented with 1 mM CaCl2 (Liao & McCallus, 1998) was concentrated by
ultrafiltration (UF) through 10 kDa molecular weight cut-off (MWCO) ultrafiltration
membrane. The UF retentate was used for hydrolytic activity experiments.
Hydrolytic activity on αs, β and κ caseins was tested by incubating 5 mg of single protein in 5
mL of Tris-HCl (20 mM, pH 6.7) with 16 µL of concentrated crude proteolytic enzyme extract
of P. fluorescens PS19. The reaction mixtures were incubated for 24, 48, 96, 120 and 144 h at 7
89
Results and discussion: Hydrolysis of casein fractions by P. fluorescens enzyme extract
°C and for 4, 8, 24, 48 and 96 h at 22 °C and aliquots taken from the mixtures, were submitted
to RP-HPLC analysis. Two controls, consisting of the peptide and the enzyme only,
respectively, were used to determine spontaneous degradation of the casein fraction or the
enzyme extract.
3.22.2 Reversed-phase – High performance liquid chromatography (RP-HPLC)
Separation of peptides in hydrolysates from P. fluorescens PS19 crude enzyme extract digesta
was performed on an RP-HPLC apparatus. This consisted of an Alliance 2695 HPLC system
(Waters, Vimodrone, Italy) equipped with a Waters 2996 diode array detector (DAD).
Hydrolysates (5–50 µL) were separated on a PLRP-S column (2.1 mm i.d. × 250 mm, 5 m,
300 Å; Polymer Laboratories Ltd, Church Stretton, Shropshire, UK) kept at 40°C. The eluents
used for the separation were: solvent A, 0.1% (v/v) trifluoroacetic acid (TFA) in MilliQ-treated
water, and solvent B, 0.1% (v/v) TFA in acetonitrile. Trifluoroacetic acid and acetonitrile were
from Thermo Scientific (Rockford, IL, USA) and Sigma–Aldrich (Milan, Italy), respectively.
The elution gradient, expressed as the solvent B proportion, was as follows: 0–5 min, 5%; 5–65
min, 55%; 65–70 min, 95%; 70–72 min, 95%, 72–76 min, 5%. The flow rate was 0.2 mL min -1,
and run-to-run time, 90 min. Absorbance was recorded at 210 nm and data were processed
using the Empower software package (Waters).
3.23 Results and discussion
In order to determine whether P. fluorescens PS19 concentrated proteolytic enzyme extract
hydrolyses αs, β and κ caseins, the substrates were incubated with enzyme in 20 mM Tris-HCl
(pH 6.7) buffer at 22 °C and 7 °C for different times. Subsequently, the digestion products were
separated by RP-HPLC.
Previous studies have been performed to understand the proteolytic activities on milk proteins
and on the casein molecules, in particular Koka and Weimer (2000) indicated that a protease
isolated from P. fluorescens RO98 preferentially hydrolysed κ-casein in artificial casein
micelles. With the same objective, Costa et al. (2002) showed that an extract of P. fluorescens
RV10 culture proteolysed κ-casein and β-casein. More recently, Nicodème (2006) characterized
an extracellular protease from Pseudomonas aureofaciens LBSA1 and identified some peptidic
bonds which had been cleaved by this enzyme on different purified casein molecules. Mu et al.
(2009) studied the proteolytic activity of Ht13 purified enzyme from raw milk-associated P.
fluorescens Rm12, and reported that the enzyme can cleave αs casein, β casein and κ casein,
obtaining after hydrolysis at least three, two and one fragments, respectively. It was generally
accepted that κ casein resides at the surface of the casein micelle as it exists naturally (Creamer,
1991; Walstra, 1990). The stability of the casein micellar system can be attributed directly to
the unique properties of κ casein. Baglinière et al. (2013), studying the destabilisation of UHT
milk due to the activity of P. fluorescens F aprX protease, found that the enzyme hydrolysed all
casein fractions with a preference for β-casein. It has been reported that metalloproteases, as
AprX, preferentially hydrolyse the κ-casein then β-casein and finally αs1 casein. Generally, this
enzyme can hydrolyse all casein molecules (Cousin, 1982; Fairbairn and Law, 1986; Koka and
Weimer, 2000).
In our study, a progressive hydrolysis of αs casein fraction [which was previously found by
casein zymography to be the most extensively hydrolysed by PS19 (data not shown)] by the P.
fluorescens PS19 concentrated enzyme extract was highlighted by HPLC. As expected, casein
degradation was higher at 22 °C and no new peptidic fragments formed when hydrolysis was
90
Results and discussion: Hydrolysis of casein fractions by P. fluorescens enzyme extract
longer than 96 h at 22 °C or 6 d at 7 °C, as reported in Fig. 3.23.1 and Fig. 3.23.2 for αs casein
fraction.
The different peptides are under identification by HPLC/MS.
Absorbance (AU210 nm )
0.27
Figure 3.23.1 Chromatographic
profiles of peptides generated from
αs-CN hydrolysis by enzymatic
extract of P. fluorescens PS19 after
4 (A), 8 (B), 24 (C) and 96 (D) h
of hydrolysis at 22 °C.
0.18
T0
0.09
A
0.00
B
C
D
-0.09
0
10
20
30
40
50
60
Absorbance (AU210 nm)
Retention time (min)
0.36
T0
0.24
A
B
0.12
C
D
0.00
E
0
10
20
30
40
50
Figure 3.23.2 Chromatographic
profiles of peptides generated
from αs-CN hydrolysis by
enzymatic
extract
of
P.
fluorescens PS19 after 24 (A), 48
(B), 96 (C), 120 (D) and 144 (E) h
of hydrolysis at 7 °C.
60
Retention time (min)
3.24 References
Baglinière F, Matéos A, Tanguy G, Jardin J, Briard-Bion V, Rousseau F, Robert B, Beaucher E,
Gaillard JL, Amiel C, Humbert G. Dary A, Gaucheron F. 2013. Proteolysis of ultra high
temperature-treated casein micelles by AprX enzyme from Pseudomonas fluorescens F induces
their destabilisation. Int Dairy J 31:55-61.
Costa M, Gomez MF, Molina LH, Simpson R, Romero A. 2002. Purification and
characterization of proteases from Pseudomonas fluorescens and their effect on milk proteins.
Arch Latinoam Nutr 52:160–166.
Cousin MA. 1982. Presence and activity of psychrotrophic microorganisms in milk and dairy
products: a review. J Food Prot 45:172–207.
Cox JM. 1993. The significance of psychrotrophic Pseudomonas in dairy products. Aust. J
Dairy Technol 4:108-113.
91
Results and discussion: Hydrolysis of casein fractions by P. fluorescens enzyme extract
Creamer LK. 1991. Some aspects of casein micelle structure. In: Parris N, Barford R (eds)
Interactions in food proteins. ACS Symp. Ser. 454. American Chemistry Society, Washington,
DC, pp 148–163.
Cromie S. 1992. Psychrotrophs and their enzyme residues in cheese milk. Aust J Dairy Technol
47:96-100.
Decimo, M., Morandi, S.,Silvetti, T., Brasca, M., 2014. Characterization of gram-negative
psychrotrophic bacteria isolated from bulk tank milk J f Food Sci doi: 10.1111/17503841.12645.
Fairbairn DJ, Law BA. 1986. Proteinases of psychrotrophic bacteria: their production,
properties, effects and control. J Dairy Res 53, 139–177.
Grufferty MB; Fox PF.1988. Milk alkaline proteinase. J Dairy Res 55:609-630.
Kohlmann KL; Nielsen SS; Steenson LR; Ladisch MR. 1991. Production of proteases by
psychrotrophic microorganisms.J Dairy Sci 74:3275-3283.
Kohlmann KL, Nielsen SS; Ladisch MR. 1991. Purification and characterization of an
extracellular protease from Pseudomonas fluorescens Md/6 grown in milk. J Dairy Sci 74:41254136.
Koka R, Weimer BC. 2000. Isolation and characterization of a protease from Pseudomonas
fluorescens RO98. J Appl Microbiol 89:280–288.
McKellar RC. 1982. Factors influencing the production of extracellular proteinase by
Pseudomonas fluorescens. J. Appl. Bacteriol. 53:305-316.
Liao CH, McCallus DE, 1998, Biochemical and genetic characterization of an extracellular
protease from Pseudomonas fluorescens CY091. Appl Environ Microbiol 64:914–921.
Mitchell GE; Ewings KN. 1985. Quantification of bacterial proteolysis causing gelation in
UHT-treated milk. N. Z. J Dairy Sci Technol 20:65-68.
Mu Z, Du M, Bai Y. 2009. Purification and properties of a heat-stable enzyme of Pseudomonas
fluorescens Rm12 from raw milk. Eur Food Res Technol 228:725–734.
Nicodème M. 2006. Identification d’une souche de Pseudomonas, bactérie psychrotrophe isolée
de lait cru. Caractérisation de sa protéase extracellulaire et des sites d’hydrolyse sur les caséines
bovines. Thesis, Université Henri Poincaré, Nancy I, France.
Sorhaug T; Stepaniak L. 1997. Psychrotrophs and their enzymes in milk and dairy products:
quality aspects. Trends Food Sci Technol 8:35-41.
Suhren G. Producer microorganisms. In Enzymes of Psychrotrophs in Raw Foods; McKellar,
RC, Ed.; CRC Press: Boca Raton, FL, 1989; pp 3-34.
Walstra P. 1990. On the stability of casein micelles. J Dairy Sci 73:1965–1979.
92
ACKNOWLEDGEMENTS
This thesis work was carried out between the years 2012 – 2014 in the Institute of Sciences of
Food Production, National Research Council in collaboration with the Department of Food,
Environmental and Nutritional Sciences at the University of Milan in Italy. Financial support
from Institute of Sciences of Food Production, National Research Council is gratefully
acknowledged.
I would like to thank my tutor prof. Ivano De Noni for giving me the opportunity to undertake
this research project.
I would like to express my immense gratitude to my co-tutor dr. Milena Brasca for giving me
the idea to do this PhD, for her continuous assistance and guidance, her precious suggestions
and encouragement that have been essential throughout my PhD study. I also thank her for
opening the doors for the Spanish collaboration.
A special thanks goes to prof. María Concepción Cabeza Briales and prof. Juan Antonio
Ordóñez Pereda for their supervision during the research work carried out in the Departament of
Nutrition, Bromatology and Technology of Food, Faculty of Veterinary, at the Complutense
University of Madrid. I am very grateful to prof. Conchita for following me every single day for
entire four months, for her enthusiasm towards the undiscovered areas, for her precise insights
and comments, for teaching me to speak the correct Spanish! I appreciate the invaluable
collaboration of dr. Raquel Velasco de Diego and dr. Carlos Santos and the help of dr. Jose
Segura Plaza with GC analysis.
I warmly thank my collegues of the Institute of Sciences of Food Production, National Research
Council for the scientific assistance and for costant support: dr. Giovanna Battelli, dr. Stefano
Morandi, dr. Tiziana Silvetti, dr. Clara Albano and Gennaro Garofalo.
I am deeply indebted to prof. Giancarlo Aldini and dr. Mara Colzani for the invaluable help in
proteomic analysis.
I wish to sincerely thank dr. Milda Stuknyte for her precious help and insightful comments on
this thesis work.
I would like to thank dr. Paolo D’Incecco, my collegue, friend, confident for his moral support
and his smiles.
I thank my beautiful family: my father Crocifisso and mother Anna, my lovely brothers
Francesco and Giuseppe and my great grandmother Pina for supporting my choices, for costant
encouragement, for all the love and for always being present.
I thank to my angel with blue eyes as mine for giving me the strength to go on and overcome
any problem.
Milan, December 2014
93
Appendix 1: Copies of abstracts of papers, oral communications and posters
APPENDIX 1. COPIES OF ABSTRACTS OF PAPERS, ORAL COMMUNICATIONS
AND POSTERS
1. Decimo M, Morandi S, Silvetti T, Brasca M. 2014. Characterization of Gram-negative
psychrotrophic bacteria isolated from bulk tank milk. Journal of Food Science doi:
10.1111/1750-3841.12645.
2. Zucali M, Bava L, Colombini S, Brasca M, Decimo M, Morandi S, Tamburini A, Crovetto
GM. 2014. Management practices and forage quality affecting the contamination of milk with
anaerobic spore-forming bacteria. Journal of the Science of Food and Agriculture doi:
10.1002/jsfa.6822.
94
Appendix 1: Copies of abstracts of papers, oral communications and posters
3. Battelli G, Silvetti T, Decimo M, Brasca M. Volatile organic compounds produced in milk
by Enterococcus faecalis. In Proceedings of the 10th International Meeting on Mountain cheese,
Dronero (CN), Italy, 14-15 September 2011, pp. 75-76 ISBN: 978-88-902754-5-6.
Abstract
Enterococci can contribute positively to the development of flavour during cheese ripening as
their presence is consistently reported in many raw milk cheeses. They can influence flavour
taste and texture of cheeses as they produce several enzymes that interact with milk
components, thus promoting important biochemical transformations. For this survey, 40
Enterococcus faecalis strains, collected in different valleys in northwest Italy, were inoculated
in milk and submitted to head-space solid-phase-micro- extraction gas chromatography-mass
spectrometry analysis. The major volatile compounds detected were: ethanol, diacetyl, acetoin,
acetic and benzoic acids. The variability was huge, demonstrating a very different enzymatic
activity among strains. Apart from other important phenological parameters for characterizing
strains that can be used as starter cultures, it seems useful to also give some information about
their capability of enhancing flavour characteristics in the cheese.
4. Decimo M, Bacterial enzymatic activities as potential markers for assessing the technological
properties of (un)processed milk. In Proceedings of the 17th Workshop on the Developments in
the Italian PhD Research on Food Science Technology and Biotechnology, University of
Bologna, Cesena, 19-21 September, 2012, pp. 299-300.
Abstract
Psychrotrophic bacteria are responsible for the highest spoilage of unprocessed or heated milk
during storage, because of their capacity to synthesize thermostable extracellular lipases and
proteinases which can hydrolyze milk fat and proteins, raising many quality and technological
problems. To date, still little is known about the presence and the hydrolytic activity of these
enzymes in milk. On these bases, this PhD thesis research project aims to acquire further
knowledge on the enzymatic activity of psychrotrophs, isolated from unprocessed milk, in order
to assess presence and type of activity of lipases and proteinases and hence to predict the overall
quality of (un)processed milk.
5. Battelli G, Bava L, Decimo M, Mattiello S, Povolo M, Sanna M, Zanini L, Sandrucci A,
Brasca M. Individuazione di modelli di aziende zootecniche per produzioni di eccellenza di
latte e derivati. In abstracts of the 3rd Dairy Meeting – AITeL, Milk and Dairy Products:
Research and Innovation. Milano, 28 September 2012.
Abstract
Il latte rappresenta uno dei principali prodotti del settore agro-alimentare lombardo. La
Lombardia detiene il primato in Italia per il latte bovino, con una produzione pari a circa il
37% di quella nazionale, essendo anche il primo produttore di formaggi, tra i quali figurano
formaggi DOP di grande pregio. Una produzione così importante per l’economia del territorio
deve avere carattere di eccellenza, un’eccellenza “globale” (microbiologica, nutraceutica e
organolettica), che non sia a scapito del benessere animale e che sia eco-sostenibile. Le
aziende che si distinguono per questi parametri di eccellenza potrebbero avvalersi di un
adeguato riconoscimento commerciale e, in prospettiva, aderire ad un Marchio di Filiera che
garantisca al consumatore non solo la qualità superiore del prodotto finito, ma anche una
gestione aziendale rispettosa dell’ambiente e degli animali. Grazie alla collaborazione di
competenze che comprendono tutta la filiera produttiva, è in corso una sperimentazione che,
coinvolgendo 30 aziende zootecniche selezionate sulla base di parametri gestionali (criteri:
95
Appendix 1: Copies of abstracts of papers, oral communications and posters
autosufficienza alimentare; quantità di insilati e/o foraggio), raggruppate in base alla
collocazione geografica (LO-MN-CO/LC), vuole identificare modelli aziendali virtuosi che
comprendono tutti i processi della filiera produttiva: la coltivazione delle essenze foraggere, la
composizione della razione, la gestione della mandria in stalla e in sala di mungitura, lo
spandimento dei liquami, la gestione del territorio, le modalità di stoccaggio del latte e la sua
trasformazione, parametri che in diversa misura differenziano le aziende nelle tre zone
considerate. Il latte prodotto da queste aziende, raggruppato nelle tre diverse zone, viene
trasformato a Grana Padano da una unico caseificio. La sperimentazione prevede sul latte di
ogni singola azienda, nella stagione estiva ed in quella invernale, analisi atte a valutarne la
qualità microbiologica, compositiva e nutrizionale. La sperimentazione prevede inoltre
l’analisi della qualità nutrizionale ed organolettica del formaggio a 9, 16 e 20 mesi.
6. Zucali M, Battelli G, Decimo M, Mattiello S, Povolo M, Sanna M, Zanini L, Guerci M,
Tamburini A, Brasca M. Qualità del latte e sostenibilità di aziende zootecniche di tre differenti
realtà lombarde. In abstracts of the 3rd Dairy Meeting – AITeL, Milk and Dairy Products:
Research and Innovation. Milano, 28 September 2012.
Abstract
Il presente lavoro si propone di studiare 3 modelli di aziende zootecniche rappresentative della
realtà lombarda (province di Como-Lecco, Lodi e Mantova) definiti in base al carico di
animali per ettaro e livello di autosufficienza alimentare, percentuale di utilizzo di alimenti
insilati nella razione, qualità e quantità di foraggio nella razione. In 30 aziende è stato valutato
l’impatto ambientale attraverso la metodologia Life Cycle Assessment, monitorato il
benessere animale, determinate le caratteristiche chimiche e microbiologiche del latte. Le
aziende di Lodi, rispetto a quelle delle altre province, sono le più grandi sia in termini di capi
allevati (128 bovine) che di ettari coltivati (55 ha), hanno acquistato più alimenti concentrati
(P<0,05) e questo ha determinato un maggior surplus di azoto a livello aziendale (P<0,005).
Sempre in queste aziende sono state rilevate maggiori percentuali di zoppie e diarree, sintomo
quest’ultimo forse dovuto all’elevata quantità di silomais in razione (34,5%). I parametri di
impatto ambientale non hanno presentato differenze significative nei tre gruppi di aziende,
anche se è possibile notare un maggior impatto delle aziende del lodigiano. Simili nelle tre
zone sono anche i contenuti di PUFA, MUFA, acidi grassi n-6 e n-3 del latte. La presenza di
elevate percentuali di fieno nella razione sembra invece aver determinato una quantità più
elevata (P<0,05) di alcuni idrocarburi quali 2-fitene, fitano, nonacosano, entriacontano e gli
esteri del fitolo nel latte delle stalle di Mantova che sono caratterizzate da una minor presenza
di silomais in razione. La carica batterica del latte di massa è rappresentata in gran parte da
batteri lattici mesofili. Ciò indica la buona qualità microbiologica del latte data dalla corretta
igiene in mungitura ed adeguata temperatura di conservazione. Nel latte delle aziende di
Mantova i batteri coliformi erano presenti in quantità più elevate rispetto alle altre zone. I
risultati ottenuti mostrano che il fattore che ha maggiormente influenzato alcuni parametri di
sostenibilità ambientale, di benessere e le caratteristiche del latte è la percentuale di inclusione
dei silomais nella razione delle bovine da latte.
7. Bava L, Zucali M, Sandrucci A, Guerci M, Battelli G, Brasca M, Povolo M, Decimo M,
Tamburini A. How different farming systems can affect nutraceutical and traceable
components of cow milk? XX Congresso Aspa – Animal Science and Production Association,
Bologna, 11-13 June, 2013.
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Appendix 1: Copies of abstracts of papers, oral communications and posters
Abstract
The aim was to analyze different dairy farming systems to clarify if the management can
influence the nutraceutical composition and traceable components of cow milk. All of the 29
farms studied were located in Northern Italy and they were members of a cheese factory which
produced Grana Padano cheese P.D.O. Information about herd composition and milk
production, feeding system and land utilization were obtained through personal interviews to
the farmers. In each farm samples of bulk milk were collected for chemical and
microbiological analyses. The farms had characteristics of high intensity: lactating cows was
on average 78.4±46.6, stocking density 4.09±2.56 LU/ha, milk production (FPCM) 27.0±4.21
kg/d cow, milk fat 3.94±0.15% and milk protein 3.43±0.15%. Most of t! he farms included
corn silage in the cow ration, forage intake was 59.5% and feed self-sufficiency was 63.6%. A
cluster analysis was performed on farm characteristics (ha of land, number of cows, milk
production level, dairy efficiency, feed self-sufficiency) and identified three different groups
of farms. Cluster 2 included 11 farms characterized by low intensity level: few lactating cows
(32.4), low stocking density (2.97 LU/ha), low corn silage intake (22.8% DMI) and high
forage intake (64.1%). Milk microbial contamination (SPC, coliform count and clostridium
spores count) was not statistically different among the groups while the lactic bacteria were
higher in milk of cluster 2 compared to the others. Nutraceutical components (CLA, MUFA,
omega 3 and omega 6 fatty acids) were similar among the clusters. Some non-volatile
hydrocarbons present in the neutral lipid fraction of milk fat (phytane, phytene, phytyl C16,
phytyl C18 unsaturated e phytyl C18 saturated) were significantly h! igher in the farms of
Cluster 2 than in Cluster 1(10 farms) e 3 (7 farms). We supposed that these molecules were
linked with the high forage content and low maize silage percentage of cows’ rations in
Cluster 2 farms. The results showed that the different level of intensity of farms involved had
a low influence on nutraceutical composition of milk and that some non-volatile hydrocarbons
in milk could be used as markers of forage composition of cow’s diet.
8. Decimo M, Bacterial enzymatic activities as potential markers for assessing the
technological properties of (un)processed milk. In Proceedings of the 18th Workshop on the
Developments in the Italian PhD Research on Food Science Technology and Biotechnology,
Universities of Padova and Udine, Conegliano, 25–27 September, 2013, pp. 239-240.
Abstract
Psychrotrophic bacteria are responsible for the highest spoilage of unprocessed or heated milk
during storage because of their capacity to synthesize thermostable extracellular proteases and
lipases. The diversity and enzymatic traits of 70 psychrotrophic strains, isolated from samples
of unprocessed milk, were investigated, and the presence of the aprX gene was ascertained.
Extracellular caseinolytic activity of three selected strains of P. fluorescens (two aprX+ and
one aprX-) was evaluated and the approximate molecular mass of their proteases was
estimated by casein zymography. Finally, the production of volatile organic compounds in
UHT milk inoculated with 5 different psychrotrophic species was evaluated by SPME-GC/MS
to investigate the relationship between the presence of these bacterial strains and the onset of
certain off flavours in milk.
9. Decimo M, Cabeza MC, Ordonez JA, Silvetti T, Morandi S, Brasca M. Caratterizzazione di
composti volatili in latte a seguito di sviluppo di batteri psicrotrofi. In abstracts of the 4th Dairy
Meeting – AITeL, Milk and Dairy Products: Research and Innovation. Padova, 12 September
2014.
97
Appendix 1: Copies of abstracts of papers, oral communications and posters
Abstract
I batteri psicrotrofi rappresentano una delle principali cause di deterioramento dei prodotti
lattiero-caseari. Tali microrganismi infatti, seppure siano facilmente distrutti dai trattamenti
termici, rilasciano enzimi termoresistenti (proteasi, lipasi e lecitinasi) in grado di influenzare le
caratteristiche di flavour, colore e texture dei prodotti e conseguentemente la loro possibilità di
conservazione.
Lo studio ha valutato i composti organici volatili (VOCs) rilasciati in latte a seguito dello
sviluppo di batteri psicrotrofi con attività lipo-proteolitica mediante microestrazione in fase
solida (SPME) accoppiata alla gas cromatografia/spettrometria di massa (GC-MS). P.
fluorescens PS14, P. fragi PS 55, P. mosselii PS39, P. rhodesiae PS62 e Serratia marcescens
S92, appartenenti alla collezione CNR-ISPA, sono stati inoculati in latte UHT, conservati a 5°C
e 10°C per 20 giorni e analizzati a tempi stabiliti.
L’analisi dei dati indica che allo sviluppo di ciascun microrganismo durante la conservazione
del latte sono associabili profili cromatografici complessi e specifici. Il latte UHT non
contaminato mostra una ridotta quantità e diversità di composti volatili rispetto al latte
inoculato; il numero di composti volatili.
10. Decimo M, Bacterial enzymatic activities as potential markers for assessing the
technological properties of (un)processed milk. In Proceedings of the 19th Workshop on the
Developments in the Italian PhD Research on Food Science Technology and Biotechnology,
University of Bari, Bari, 24-26 September, 2014, pp. 278-282.
Abstract
Psychrotrophic bacteria are responsible for the highest spoilage of unprocessed or heated milk
during storage because of their capacity to synthesize thermostable extracellular proteases and
lipases leading to gelation, off-odours/flavours, loss of sensory quality and shelf life. This PhD
thesis dealt with the assessment of the enzymatic traits of 80 raw milk-associated
psychrotrophic bacteria and the detection of specific molecules and metabolites derived from
bacterial enzymatic degradation of milk components that could be used as a good markers to
early recognize the activity for psychrotrophic strains in unprocessed milk.
Quaderni della Ricerca
1 De Noni I, Brasca M, Battelli G, Cattaneo S, Decimo M, Masotti F, Morandi S, Pellegrino L,
Ranghetti A, Silvetti T, Stuknyte M. (2013) Silter: Starter autoctoni e tipicità. Regione
Lombardia - Quaderni della ricerca n. 156.
2 Brasca M, Bava L, Zucali M, Tamburini A, Guerci M, Sandrucci A, Mattiello S, Andreoli E,
Battini M, Decimo M, Morandi S, Battelli G, Povolo M, Pellizzola V, Passolungo L, Sanna M,
Zanini L. (2014) Modelli di azienda zootecnica per latte e formaggi d’eccellenza. – Regione
Lombardia – Quaderni della ricerca n. 163.
98
Appendix 2: Index of tables
APPENDIX 2. INDEX OF TABLES
Table 3.3.1 Phenotypic properties of 63 Pseudomonas strains isolated from raw milk
Table 3.3.2 Phenotypic properties of 17 Enterobacteriaceae strains isolated from raw
milk
Table 3.3.3 An overview of Pseudomonas strains: enzymatic characterization at 7, 22
and 30 °C and detection of aprX gene
Table 3.3.4 An overview of Enterobacteriaceae strains: enzymatic characterization at
7, 22 and 30 °C and detection of aprX gene
Table 3.8.1 VOCs detected in UHT milk at T0 and during storage time (days) at 10 °C
and 5 °C
Table 3.8.2 VOCs detection in spoiled UHT milk during storage time (days) at 10 °C
Table 3.8.3 VOCs detected in spoiled UHT milk at 5°C after 20 days of storage
Table 3.13.1 Concentrations of free fatty acids (mg/100 g total lipid) in control and
inoculated samples after incubation at 30°C for 1 and 4 days by four selected
psychrotrophic strains
99
33
35
37
40
50
53
56
73
Appendix 3: Index of figures
APPENDIX 3. INDEX OF FIGURES
Figure 3.3.1 Dendrogram derived from profiles by the Random Amplification of
Polymorphic DNA (RAPD-PCR) generated with primers M13 and OPAA-10 of the
80 GNP strains isolated from different raw milk samples. The profile grouping was
done with the BioNumeric 5.0 software package, using the unweighted pair group
method with arithmetic averages (UPGMA) cluster analysis.
Figure 3.8.1 SPME-GC/MS chromatogram of UHT milk contaminated with P.
fluorescens PS14. Peaks: (1) acetone; (2) dimethylsulphide; (3) hexane; (4) 2,3
butanedione; (5) 2-butanone; (6) 2-pentanone; (7) pentanal; (8) mercaptoacetone, (9)
3-methylbutanol;(10) disulphide dimethyl; (11) butanoic acid, (12) hexanal; (13)
dimethyl sulfoxide; (14) 2-heptanone; (15) heptanal; (16) dimethyl sulfone; (17)
hexanoic acid; (18) 5-hepten-2one-6-methyl; (19) benzaldehyde; (20) octanal;(21) 2nonanone; (22) octanoic acid.
Figure 3.13.1 Specificity of lipases from psychrotrophic strains toward tributyrin
TAG
Figure 3.13.2 Specificity of lipases from psychrotrophic strains toward tricaproin
TAG
Figure 3.13.3 Specificity of lipases from psychrotrophic strains toward trimyristin
TAG
Figure 3.13.4 Specificity of lipases from psychrotrophic strains toward triolein TAG
Figure 3.13.5 Total FFAs concentration (mg/g total lipid) in control and inoculated
samples after incubation at 30 °C for 24 h and 4 days
Figure 3.18.1 Extracellular caseinolytic activity of P. fluorescens strains: casein
zymography under non reducing conditions
Figure 3.18.2 Extracellular caseinolytic activity of P. fluorescens strain PS19: casein
zymography under non reducing conditions of concentrated proteolytic enzyme
extract with A) 0,1% of sodium caseinate; B) 0,1% of αs – casein; C) 0,1% of β –
casein and D) 0,1% of κ – casein
Figure 3.18.3 ClustalW sequence alignment of protein identified by proteomic
analysis of proteolytic 45 kDa band from P. fluorescens PS19. Peptides as retrivied by
mass spectrometry are shown in bold. Symbols (.), (:) and (*) represent the degree of
conservation within the compared sequences
Figure 3.18.4 ClustalW sequence alignment of proteins identified by proteomic
analysis of P. fluorescens PS19 concentrated (UF 10 kDa) proteolytic extract (in
solution digest) and proteolytic band of 15 kDa. Peptide coverage is indicated by
highlighted characters: PS19-Trypsin retentate (shown in bold), PS19-Trypsin 15 kDa
(underlined and shown in bold)
Figure 3.23.1 Chromatographic profiles of peptides generated from αs-CN hydrolysis
by enzymatic extract of P. fluorescens PS19 after 4 (A), 8 (B), 24 (C) and 96 (D) h of
hydrolysis at 22 °C
Figure 3.23.2 Chromatographic profiles of peptides generated from αs-CN hydrolysis
by enzymatic extract of P. fluorescens PS19 after 24 (A), 48 (B), 96 (C), 120 (D) and
144 (E) h of hydrolysis at 7 °C
100
32
48
69
69
70
70
71
82
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85
91
91